Compounds for Increasing Lipid Synthesis and Storage

ABSTRACT

This invention relates to methods for increasing lipid accumulation and lipid production in cells. Methods of producing biofuel from cells and preparing nutraceuticals comprising lipids produced according to a method provided herein are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No. 15/138,066 filed Apr. 25, 2016, which claims the benefit of U.S. Provisional Application Ser. No. 62/152,717, filed Apr. 24, 2015, the disclosure of which is incorporated herein by reference in its entirety.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under Grant Nos. EPS-1004094 and 1264409, awarded by the National Science Foundation. The Government has certain rights in the invention.

TECHNICAL FIELD

This invention relates to methods for increasing lipid accumulation and lipid production in cells.

BACKGROUND

Algae are a diverse group of microorganisms that include species that inhabit marine, freshwater and terrestrial niches. It is estimated that 30-50% of the net photosynthetic productivity on plant earth is contributed by phytoplankton including algae. Algae may also be employed for the production of biofuels, and can be grown in poor quality water and under a large variety of environmental conditions (see, e.g., Singh et al., Bioresource Technology, 2011, 102, 26-34 and Hu et al., The Plant Journal, 2008, 54, 621-639). Additionally, during photosynthesis algae fix CO₂ into biomass thus addressing concerns about the generation of carbon emissions.

SUMMARY

The present application provides, inter alia, a method of increasing lipid accumulation in a cell, comprising contacting the cell with an effective amount of a compound of Formula Ia:

or a pharmaceutically acceptable salt thereof, wherein:

L is selected from the group consisting of a bond, C₁₋₄ alkylene, C₁₋₄ hydroxyalkylene, C₁₋₄ alkyleneoxy, —(C₁₋₄ alkylene)-NR^(L)—(C₁₋₄ alkylene)_(n)-, C(═O), and C(═O)NH;

R^(L) is selected from the group consisting of H and methyl;

R³ is H or C₁₋₄ alkoxy;

R⁴ is selected from the group consisting of H and piperidinyl;

Cy¹ is selected from the group consisting of phenyl, C₃₋₈ cycloalkyl, 4-6 membered heterocycloalkyl, and 4-10 membered heteroaryl;

R^(Cy) is selected from the group consisting of halo, C₁₋₄ alkyl, C₁₋₄ alkoxy, phenyl, —(C₁₋₄ alkylene)-(phenyl), C₃₋₁₀ cycloalkyl, and —(C₁₋₄ alkylene)-(C₃₋₁₀ cyloalkyl), wherein each phenyl and the phenyl group of —(C₁₋₄ alkylene)-(phenyl) is optionally substituted by 1 or 2 substituents independently selected from the group consisting of OH, halo, C₁₋₄ alkyl, and C₁₋₄ alkoxy;

n is 0 or 1; and

p is 0, 1, 2, 3, 4, or 5; or

a compound of Formula Ib:

or a pharmaceutically acceptable salt thereof, wherein:

L is selected from the group consisting of a bond, C₁₋₄ alkylene, C₁₋₄ hydroxyalkylene, and C(═O);

R⁴ is selected from the group consisting of H and piperidinyl; and

R^(Cy) is selected from the group consisting of C₁₋₄ alkyl, phenyl, —(C₁₋₄ alkylene)-(phenyl), C₃₋₁₀ cycloalkyl, and —(C₁₋₄ alkylene)-(C₃₋₁₀ cyloalkyl), wherein each phenyl and the phenyl group of —(C₁₋₄ alkylene)-(phenyl) is optionally substituted by 1 or 2 substituents independently selected from the group consisting of OH, halo, C₁₋₄ alkyl, and C₁₋₄ alkoxy; or

a compound of Formula Ic:

or a pharmaceutically acceptable salt thereof, wherein:

L³ is selected from the group consisting of a bond and C(O)NH;

R^(3a) is selected from the group consisting of H, C₁₋₄ alkyl, C₁₋₄ alkoxy, -phenyl-(C₁₋₄ alkylene)-R^(b), -(phenyl)-SO₂R^(b), and 5-6 membered heteroaryl, wherein the 5-6 membered heteroaryl is optionally substituted with a C₁₋₄ alkyl group;

R^(b) is selected from the group consisting of 5-6 membered heteroaryl and 5-6 membered heterocycloalkyl, wherein the 5-6 membered heterocycloalkyl is optionally substituted by a C₁₋₄ alkyl group;

L⁴ is selected from the group consisting of a bond, S, C(═O)NH, C₁₋₆ alkyleneoxy, and SO₂-Cy²-Cy³;

Cy² and Cy³ are each an independently selected 5-6 membered heterocycloalkyl group; and

R^(4a) is selected from the group consisting of H, C₁₋₄ alkyl, —(C₁₋₄ alkylene)-(C₃₋₆ cycloalkyl), 5-6 membered heterocyloalkyl, and —(C₁₋₄ alkylene)-(phenyl); or

a compound of Formula IIa:

or a pharmaceutically acceptable salt thereof, wherein:

L¹ is selected from the group consisting of a bond, C₁₋₄ alkylene, C₂₋₄ alkenylene, C₁₋₄ hydroxyalkylene, C₁₋₄ alkyleneoxy, 4-6 membered heterocycloalkylene, and C(═O), wherein the C₁₋₄ alkyleneoxy is further optionally substituted by one OH group;

R^(1a) is selected from the group consisting of OH, C₁₋₄ alkyl, C₅₋₁₀ cycloalkyl, 8-10 membered heteroaryl, phenyl, —(C₁₋₄ alkylene)-(phenyl), and 10-13 membered heteroaryl, wherein the C₅₋₁₀ cycloalkyl, phenyl, and the phenyl group of the —(C₁₋₄ alkylene)-(phenyl) are each optionally substituted by 1, 2, or 3 substituents independently selected from the group consisting of halo, OH, NO₂, C₁₋₄ alkyl, and C₁₋₄ alkoxy, and wherein the 10-13 membered heteroaryl is optionally substituted by one halo group;

R² is selected from the group consisting of H, halo, OH, NO₂, C₁₋₄ alkyl, C₁₋₄ alkoxy, and —(C₁₋₄ alkylene)-(8-10 membered heteroaryl);

R³ is selected from the group consisting of H, halo, C₁₋₄ alkoxy, and phenoxy; or alternatively, R² and R³ come together, in combination with the carbon atoms to which they are attached, to form a 5-6 membered heterocycloalkyl or a C₆₋₁₀ aryl;

R⁴ is selected from the group consisting of H, OH, and C₁₋₄ alkoxy;

R⁵ is selected from the group consisting of H, halo, C₁₋₄ alkoxy, and phenoxy; and

R⁶ is selected from the group consisting of H and oxo; or

a compound of Formula IIIa:

or a pharmaceutically acceptable salt thereof, wherein:

L¹ is selected from the group consisting of a bond, NH, N(C₁₋₄ alkyl), C₂₋₄ alkynyl, NHC(═O)(C₁₋₄ thioalkyl), C₁₋₆ alkyleneoxy, 5-6 membered heterocycloalkyl, NHC(═O)C₁₋₄ alkylene, and (C₁₋₄ alkylene)-NHC(═O)—(C₁₋₄ alkylene)-, wherein the C₁₋₆ alkyleneoxy is further optionally substituted by one OH group;

R^(1a) is selected from the group consisting of H, C₁₋₄ alkyl, C₂₋₄ alkenyl, C₁₋₄ cyanoalkylene, 5-6 membered heterocycloalkyl, 5-6 membered heteroaryl, NH(C₁₋₄ alkyl), and NH(C₂₋₄ alkenyl), wherein the C₁₋₄ alkyl and C₂₋₄ alkenyl are each optionally substituted by one substituent selected from the group consisting of phenyl and hydroxyphenyl, and wherein the 5-6 membered heterocycloalkyl and 5-6 membered heteroaryl are each optionally substituted by 1 or 2 substituents independently selected from the group consisting of C₁₋₄ alkyl, NH₂ and di-halo substituted phenyl;

L² is selected from the group consisting of a bond and 5-6 membered heterocycloalkyl; and

R^(2a) is selected from the group consisting of H, C₁₋₄ hydroxyalkyl, —SO₂(C₁₋₄ alkyl).

In some embodiments, the compound is a compound of Formula Ia. In some embodiments, the compound is a compound of Formula II. In some embodiments, the compound is a compound of Formula IIIa.

In some embodiments, the cell is a eukaryotic cell or a cyanobacteria. In some embodiments, the cell is selected from the group consisting of an algal cell, a drosophila cell, a yeast cell, or a fungus cell. In some embodiments, the cell is an algal cell. In some embodiments, the cell is selected from the group consisting of:

Chlamydomonas reinhardtii;

Chlorella sorokiniana;

Tetrachlorella alterans;

C. protothecoides;

C. vulgaris; and

Nannochloropsis sp.

In some embodiments, the increasing lipid accumulation comprises increasing fatty acid accumulation. In some embodiments, the increasing lipid accumulation comprises increasing polyunsaturated fatty acid accumulation. In some embodiments, the increasing lipid accumulation comprises increasing triacylglycerol accumulation. In some embodiments, the increasing lipid accumulation comprises increasing fatty acid accumulation and increasing triacylglycerol accumulation. In some embodiments, the increasing lipid accumulation comprises increasing polyunsaturated fatty acid accumulation and increasing triacylglycerol accumulation.

In some embodiments, the method further comprises increasing carbohydrate accumulation in the cell. In some embodiments, the method further comprises increasing carbohydrate production in the cell.

In some embodiments, the lipid accumulation is increased by greater than about 1.5-fold as compared to a cell that has not been contacted by the compound. In some embodiments, the lipid accumulation is increased by greater than about 2-fold as compared to a cell that has not been contacted by the compound. In some embodiments, the lipid accumulation is increased by greater than about 2.5-fold as compared to a cell that has not been contacted by the compound.

In some embodiments, the contacting the cell with the compound does not reduce the rate of cellular growth compared to a cell that has not been contacted by the compound. In some embodiments, contacting the cell with the compound does not reduce the rate of photosynthesis of the cell compared to a cell that has not been contacted by the compound. In some embodiments, the contacting does not adversely affect the viability of the cell.

In some embodiments, the lipid accumulation is increased without exposing the cell to environmental stress. In some embodiments, the environmental stress comprises nutrient deprivation. In some embodiments, the environmental stress comprises depriving the cell of nitrogen, depriving the cell of sulfur, depriving the cell of metal, or any combination thereof. In some embodiments, the environmental stress comprises depriving the cell of nitrogen, depriving the cell of sulfur, depriving the cell of metal, or any combination thereof.

The present application further provides a method of increasing lipid production in a cell, comprising contacting the cell with an effective amount of a compound of

or a pharmaceutically acceptable salt thereof, wherein:

L is selected from the group consisting of a bond, C₁₋₄ alkylene, C₁₋₄ hydroxyalkylene, C₁₋₄ alkyleneoxy, —(C₁₋₄ alkylene)-NR^(L)—(C₁₋₄ alkylene)_(n)-, C(═O), and C(═O)NH;

R^(L) is selected from the group consisting of H and methyl;

R³ is H or C₁₋₄ alkoxy;

R⁴ is selected from the group consisting of H and piperidinyl;

Cy¹ is selected from the group consisting of phenyl, C₃₋₈ cycloalkyl, 4-6 membered heterocycloalkyl, and 4-10 membered heteroaryl;

R^(Cy) is selected from the group consisting of halo, C₁₋₄ alkyl, C₁₋₄ alkoxy, phenyl, —(C₁₋₄ alkylene)-(phenyl), C₃₋₁₀ cycloalkyl, and —(C₁₋₄ alkylene)-(C₃₋₁₀ cyloalkyl), wherein each phenyl and the phenyl group of —(C₁₋₄ alkylene)-(phenyl) is optionally substituted by 1 or 2 substituents independently selected from the group consisting of OH, halo, C₁₋₄ alkyl, and C₁₋₄ alkoxy;

n is 0 or 1; and

p is 0, 1, 2, 3, 4, or 5; or

a compound of Formula Ib:

or a pharmaceutically acceptable salt thereof, wherein:

L is selected from the group consisting of a bond, C₁₋₄ alkylene, C₁₋₄ hydroxyalkylene, and C(═O);

R⁴ is selected from the group consisting of H and piperidinyl; and

R^(Cy) is selected from the group consisting of C₁₋₄ alkyl, phenyl, —(C₁₋₄ alkylene)-(phenyl), C₃₋₁₀ cycloalkyl, and —(C₁₋₄ alkylene)-(C₃₋₁₀ cyloalkyl), wherein each phenyl and the phenyl group of —(C₁₋₄ alkylene)-(phenyl) is optionally substituted by 1 or 2 substituents independently selected from the group consisting of OH, halo, C₁₋₄ alkyl, and C₁₋₄ alkoxy; or

a compound of Formula Ic:

or a pharmaceutically acceptable salt thereof, wherein:

L³ is selected from the group consisting of a bond and C(O)NH;

R^(3a) is selected from the group consisting of H, C₁₋₄ alkyl, C₁₋₄ alkoxy, -phenyl-(C₁₋₄ alkylene)-R^(b), -(phenyl)-SO₂R^(b), and 5-6 membered heteroaryl, wherein the 5-6 membered heteroaryl is optionally substituted with a C₁₋₄ alkyl group;

R^(b) is selected from the group consisting of 5-6 membered heteroaryl and 5-6 membered heterocycloalkyl, wherein the 5-6 membered heterocycloalkyl is optionally substituted by a C₁₋₄ alkyl group;

L⁴ is selected from the group consisting of a bond, S, C(═O)NH, C₁₋₆ alkyleneoxy, and SO₂-Cy²-Cy³;

Cy² and Cy³ are each an independently selected 5-6 membered heterocycloalkyl group; and

R^(4a) is selected from the group consisting of H, C₁₋₄ alkyl, —(C₁₋₄ alkylene)-(C₃₋₆ cycloalkyl), 5-6 membered heterocyloalkyl, and —(C₁₋₄ alkylene)-(phenyl); or

a compound of Formula IIa:

or a pharmaceutically acceptable salt thereof, wherein:

L¹ is selected from the group consisting of a bond, C₁₋₄ alkylene, C₂₋₄ alkenylene, C₁₋₄ hydroxyalkylene, C₁₋₄ alkyleneoxy, 4-6 membered heterocycloalkylene, and C(═O), wherein the C₁₋₄ alkyleneoxy is optionally further substituted by one OH group;

R^(1a) is selected from the group consisting of OH, C₁₋₄ alkyl, C₅₋₁₀ cycloalkyl, 8-10 membered heteroaryl, phenyl, —(C₁₋₄ alkylene)-(phenyl), and 10-13 membered heteroaryl, wherein the C₅₋₁₀ cycloalkyl, phenyl, and the phenyl group of the —(C₁₋₄ alkylene)-(phenyl) are each optionally substituted by 1, 2, or 3 substituents independently selected from the group consisting of halo, OH, NO₂, C₁₋₄ alkyl, and C₁₋₄ alkoxy, and wherein the 10-13 membered heteroaryl is optionally substituted by one halo group;

R² is selected from the group consisting of H, halo, OH, NO₂, C₁₋₄ alkyl, C₁₋₄ alkoxy, and —(C₁₋₄ alkylene)-(8-10 membered heteroaryl);

R³ is selected from the group consisting of H, halo, C₁₋₄ alkoxy, and phenoxy; or

alternatively, R² and R³ come together, in combination with the carbon atoms to which they are attached, to form a 5-6 membered heterocycloalkyl or a C₆₋₁₀ aryl;

R⁴ is selected from the group consisting of H, OH, and C₁₋₄ alkoxy;

R⁵ is selected from the group consisting of H, halo, C₁₋₄ alkoxy, and phenoxy; and

R⁶ is selected from the group consisting of H and oxo; or

a compound of Formula IIIa:

or a pharmaceutically acceptable salt thereof, wherein:

L¹ is selected from the group consisting of a bond, NH, N(C₁₋₄ alkyl), C₂₋₄ alkynyl, NHC(═O)(C₁₋₄ thioalkyl), C₁₋₆ alkyleneoxy, 5-6 membered heterocycloalkyl, NHC(═O)C₁₋₄ alkylene, and (C₁₋₄ alkylene)-NHC(═O)-(C₁₋₄ alkylene)-, wherein the C₁₋₆ alkyleneoxy is further optionally substituted by one OH group;

R^(1a) is selected from the group consisting of H, C₁₋₄ alkyl, C₂₋₄ alkenyl, C₁₋₄ cyanoalkylene, 5-6 membered heterocycloalkyl, 5-6 membered heteroaryl, NH(C₁₋₄ alkyl), and NH(C₂₋₄ alkenyl), wherein the C₁₋₄ alkyl and C₂₋₄ alkenyl are each optionally substituted by one substituent selected from the group consisting of phenyl and hydroxyphenyl, and wherein the 5-6 membered heterocycloalkyl and 5-6 membered heteroaryl are each optionally substituted by 1 or 2 substituents independently selected from the group consisting of C₁₋₄ alkyl, NH₂ and di-halo substituted phenyl;

L² is selected from the group consisting of a bond and 5-6 membered heterocycloalkyl; and

R^(2a) is selected from the group consisting of H, C₁₋₄ hydroxyalkyl, —SO₂(C₁₋₄ alkyl).

In some embodiments, the cell is a eukaryotic cell or a cyanobacteria. In some embodiments, the cell is selected from the group consisting of an algal cell, a drosophila cell, a yeast cell, or a fungus cell. In some embodiments, the cell is an algal cell. In some embodiments, the cell is selected from the group consisting of:

Chlamydomonas reinhardtii;

Chlorella sorokiniana;

Tetrachlorella alterans;

C. protothecoides;

C. vulgaris; and

Nannochloropsis sp.

In some embodiments, the increasing lipid production comprises increasing fatty acid production. In some embodiments, the increasing lipid production comprises increasing polyunsaturated fatty acid production. In some embodiments, the increasing lipid production comprises increasing triacylglycerol production. In some embodiments, the increasing lipid production comprises increasing fatty acid production and increasing triacylglycerol production. In some embodiments, the increasing lipid production comprises increasing polyunsaturated fatty acid production and increasing triacylglycerol production.

In some embodiments, the method further comprises increasing carbohydrate accumulation in the cell. In some embodiments, the method further comprises increasing carbohydrate production in the cell.

In some embodiments, the lipid production is increased by greater than about 1.5 fold as compared to a cell that has not been contacted by the compound. In some embodiments, the lipid production is increased by greater than about 2 fold as compared to a cell that has not been contacted by the compound. In some embodiments, the lipid production is increased by greater than about 2.5 fold as compared to a cell that has not been contacted by the compound.

In some embodiments, the contacting the cell with the compound does not reduce the rate of cellular growth compared to a cell that has not been contacted by the compound. In some embodiments, contacting the cell with the compound does not reduce the rate of photosynthesis of the cell compared to a cell that has not been contacted by the compound. In some embodiments, the contacting does not adversely affect the viability of the cell.

In some embodiments, the lipid production is increased without exposing the cell to environmental stress. In some embodiments, the environmental stress comprises nutrient deprivation. In some embodiments, the environmental stress comprises depriving the cell of nitrogen, depriving the cell of sulfur, depriving the cell of metal, or any combination thereof. In some embodiments, the environmental stress comprises depriving the cell of nitrogen, depriving the cell of sulfur, depriving the cell of metal, or any combination thereof.

The present application further provides a method of increasing lipid accumulation in a cell, comprising contacting the cell with an effective amount of a compound selected from the group of compounds provided in Table 1A, Table 1B, Table 1C, Table 2A, Table 3A, Table 4, Table 5, and Table 6, or a pharmaceutically acceptable salt thereof.

The present application further provides a method of increasing lipid production in a cell, comprising contacting the cell with an effective amount of a compound selected from the group provided in Table 1A, Table 1B, Table 1C, Table 2A, Table 3A, Table 4, Table 5, and Table 6, or a pharmaceutically acceptable salt thereof.

The present application further provides a method of increasing lipid accumulation in a cell, comprising contacting the cell with an effective amount of a compound selected from the group provided in Table 4, Table 5, and Table 6, or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is selected from the group consisting of:

(2-(3,4-dimethoxyphenyl)-N-methyl-N-(4-nitrobenzyl)ethan-1-amine);

(4-((4-(2-methoxyphenyl)piperazin-1-yl)methyl)phenol); and

(3-(adamantan-1-yl(methyl)amino)propanenitrile;

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is selected from the group consisting of:

(2-(3,4-dimethoxyphenyl)-N-methyl-N-(4-nitrobenzyl)ethan-1-amine);

(4-((4-(2-methoxyphenyl)piperazin-1-yl)methyl)phenol); and

(3-(adamantan-1-yl(methyl)amino)propanenitrile hydrochloride).

In some embodiments, the cell is a eukaryotic cell or a cyanobacteria. In some embodiments, the cell is selected from the group consisting of an algal cell, a drosophila cell, a yeast cell, or a fungus cell. In some embodiments, the cell is an algal cell. In some embodiments, the cell is selected from the group consisting of:

Chlamydomonas reinhardtii;

Chlorella sorokiniana;

Tetrachlorella alterans;

C. protothecoides;

C. vulgaris; and

Nannochloropsis sp.

In some embodiments, the increasing lipid accumulation comprises increasing fatty acid accumulation. In some embodiments, the increasing lipid accumulation comprises increasing polyunsaturated fatty acid accumulation. In some embodiments, the increasing lipid accumulation comprises increasing triacylglycerol accumulation. In some embodiments, the increasing lipid accumulation comprises increasing fatty acid accumulation and increasing triacylglycerol accumulation. In some embodiments, the increasing lipid accumulation comprises increasing polyunsaturated fatty acid accumulation and increasing triacylglycerol accumulation.

In some embodiments, the method further comprises increasing carbohydrate accumulation in the cell. In some embodiments, the method further comprises increasing carbohydrate production in the cell.

In some embodiments, the lipid accumulation is increased by greater than about 1.5 fold as compared to a cell that has not been contacted by the compound. In some embodiments, the lipid accumulation is increased by greater than about 2 fold as compared to a cell that has not been contacted by the compound. In some embodiments, the lipid accumulation is increased by greater than about 2.5 fold as compared to a cell that has not been contacted by the compound.

In some embodiments, the contacting the cell with the compound does not reduce the rate of cellular growth compared to a cell that has not been contacted by the compound.

In some embodiments, contacting the cell with the compound does not reduce the rate of photosynthesis of the cell compared to a cell that has not been contacted by the compound. In some embodiments, the contacting does not adversely affect the viability of the cell. In some embodiments, the lipid accumulation is increased without exposing the cell to environmental stress. In some embodiments, the environmental stress comprises nutrient deprivation. In some embodiments, the environmental stress comprises depriving the cell of nitrogen, depriving the cell of sulfur, depriving the cell of metal, or any combination thereof.

The present application further provides a method of increasing lipid production in a cell, comprising contacting the cell with an effective amount of a compound selected from the group provided in Table 4, Table 5, and Table 6, or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is selected from the group consisting of:

(2-(3,4-dimethoxyphenyl)-N-methyl-N-(4-nitrobenzyl)ethan-1-amine);

(4-((4-(2-methoxyphenyl)piperazin-1-yl)methyl)phenol); and

(3-(adamantan-1-yl(methyl)amino)propanenitrile;

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is selected from the group consisting of:

(2-(3,4-dimethoxyphenyl)-N-methyl-N-(4-nitrobenzyl)ethan-1-amine);

(4-((4-(2-methoxyphenyl)piperazin-1-yl)methyl)phenol); and

(3-(adamantan-1-yl(methyl)amino)propanenitrile hydrochloride).

In some embodiments, the cell is a eukaryotic cell or a cyanobacteria. In some embodiments, the cell is selected from the group consisting of an algal cell, a drosophila cell, a yeast cell, or a fungus cell. In some embodiments, the cell is an algal cell. In some embodiments, the cell is selected from the group consisting of:

Chlamydomonas reinhardtii;

Chlorella sorokiniana;

Tetrachlorella alterans;

C. protothecoides;

C. vulgaris; and

Nannochloropsis sp.

In some embodiments, the increasing lipid production comprises increasing fatty acid production. In some embodiments, the increasing lipid production comprises increasing polyunsaturated fatty acid production. In some embodiments, the increasing lipid production comprises increasing triacylglycerol production. In some embodiments, the increasing lipid production comprises increasing fatty acid production and increasing triacylglycerol production. In some embodiments, the increasing lipid production comprises increasing polyunsaturated fatty acid production and increasing triacylglycerol production.

In some embodiments, the method further comprises increasing carbohydrate accumulation in the cell. In some embodiments, the method further comprises increasing carbohydrate production in the cell.

In some embodiments, the lipid production is increased by greater than about 1.5 fold as compared to a cell that has not been contacted by the compound. In some embodiments, the lipid production is increased by greater than about 2 fold as compared to a cell that has not been contacted by the compound. In some embodiments, the lipid production is increased by greater than about 2.5 fold as compared to a cell that has not been contacted by the compound.

In some embodiments, the contacting the cell with the compound does not reduce the rate of cellular growth compared to a cell that has not been contacted by the compound. In some embodiments, contacting the cell with the compound does not reduce the rate of photosynthesis of the cell compared to a cell that has not been contacted by the compound. In some embodiments, the contacting does not adversely affect the viability of the cell.

In some embodiments, the lipid production is increased without exposing the cell to environmental stress. In some embodiments, the environmental stress comprises nutrient deprivation. In some embodiments, the environmental stress comprises depriving the cell of nitrogen, depriving the cell of sulfur, depriving the cell of metal, or any combination thereof.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

DESCRIPTION OF DRAWINGS

FIG. 1 shows EC₅₀ (μM) values and microscopy images of compounds 1, 62, and 136 in Chlamydomonas reinhardtii cells.

FIGS. 2A-2C show algal cell growth over a period of 72 hours in the presence of compound 1 (FIG. 2A), compound 62 (FIG. 2B), and compound 136 (FIG. 2C).

FIG. 3 shows increased lipid accumulation in cells in the presence of compounds 1, 62, and 136.

FIG. 4 shows increased lipid accumulation in cells over 72 hours in the presence of compounds 1, 62, and 136.

FIG. 5 shows increased fatty acid levels in cells in the presence of compounds 1, 62, and 136.

FIG. 6 shows increased triacylglyercol levels in cells in the presence of compounds 1, 62, and 136.

FIG. 7 shows the levels of chlorophyll A (Chl A), chlorophyll B (Chl B) and carotenoid in cells in the presence of compounds 1, 62, and 136.

FIG. 8 shows the cellular effects of compounds 1, 62, and 136 on the levels of proteins required for photosynthesis.

FIG. 9 shows increased carbohydrate levels in cells in the presence of compounds 1, 62, and 136.

FIG. 10 shows the effect of compounds 1, 62, and 136 on the total level of proteins in cells.

FIGS. 11A-11C show growth of various algal species in the presence of compound 1 (FIG. 11A), compound 62 (FIG. 11B), and compound 136 (FIG. 11C).

FIGS. 12A-12C show lipid body accumulation in C. reinhardtii cells using representative compounds of Formula Ia (FIG. 12A), Formula IIa (FIG. 12B), and Formula IIIa (FIG. 12C). Nile red fold-change values in the primary screening experiment at 10 μM final compound concentration (i) and Nile red fold-change values in the confirmatory screen at 10 μM compound concentration (ii) are provided for each compound. The scale bar of each image represents 5 μm.

DETAILED DESCRIPTION

There are several advantages to employing algae for the production of biofuels, for example, high productivity due to rapid growth and the ability to accumulate lipid to 20-40% dry weight and potentially 100-fold more oil per acre than soybeans or other oil-seed bearing plants. However, at the present time algal oils are not ready for commercialization due to high costs in producing and processing sufficient biomass given the primitive state of knowledge of algal biology and biotechnology (see, e.g., Singh et al., Bioresource Technology, 2011, 102, 26-34 and Hannon et al., Biofuels, 2010, 1, 763-784).

Algae are a diverse group of microorganisms that include species that inhabit marine, freshwater and terrestrial niches. It is estimated that 30-50% of the net photosynthetic productivity on plant earth is contributed by phytoplankton including algae. Yet, our understanding of the biology, metabolism and genetics of any algal species is limited. Among the microalga, Chlamydomonas reinhardtii, is widely employed as a model organism. Chlamydomonas is capable of harnessing light and fixing CO₂ to produce structural and energy rich storage compounds, including carbohydrates, lipids, and proteins. Desirable characteristics for use of algae as a biofuel feedstock include: rapid growth rate, high lipid product content, tolerance to variable environmental conditions, such as poor water quality, high salinity, extremes of pH, resistance to pathogens, ability to compete for micronutrients, and ease of harvesting and extraction (see e.g., Hannon et al., Biofuels, 2010, 1, 763-784, Chisti, Y. Biotechnology Advances, 2007, 25, 294-306, and Deng et al., Biomass and Bioenergy, 2011, 35, 1811-1817). All of these factors may not be realized in one algal species but understanding the factors that enhance or limit, for example, biomass accumulation in a model organism such as Chlamydomonas will ultimately increase our ability to select and/or generate strains with enhanced traits useful for biofuel production.

Conditions currently employed to induce lipid accumulation in algae require an environmental stress, particularly nutrient deprivation of nitrogen, sulphur, or some metal (see, e.g., Deng et al., Biomass and Bioenergy, 2011, 35, 1811-1817; Gamier et al., Journal of Proteomics, 2014, 105, 107-120; and Msanne et al., Phytochemistry, 2012, 75, 50-59). Nutrient limitation may be achieved during normal growth when cultures reach saturation density. For example, nitrogen may become limiting at this time and triglyceride rich lipid droplets will become visible and measurable (see e.g, Msanne et al., Phytochemistry, 2012, 75, 50-59 and Wase et al., Journal of Proteome Research, 2014, 13, 1373-1396). However, this may be commensurate with cessation of protein synthesis, bleaching of the cultures as the chlorophylls are degraded, depletion of the photosynthetic enzymes such as RuBisCO, and turnover of membrane lipids, including thylakoid lipids (see, e.g., Wase et al., Journal of Proteome Research, 2014, 13, 1373-1396). What is desired are methods to activate metabolic pathways leading to storage of lipids without diversion of carbon and nitrogen away from other anabolic processes required for cell growth and division.

Accordingly, the present application provides methods of increasing lipid accumulation and production in cells (e.g., algal cells) using activators of lipid synthesis and storage provided herein.

Compounds

The present application provides compounds that are activators of lipid synthesis and lipid storage in cells. In some embodiments, the compound is a compound of Formula I:

or a pharmaceutically acceptable salt thereof, wherein:

substituents R¹, R², R³, R⁴, and R⁵ of Formula I are as defined herein.

In some embodiments, a compound of Formula I is substituted according to the substituents provided in Table 1. In some embodiments, each R¹, R², R³, R⁴, and R⁵, unless specified otherwise in Table 1, is selected from the group consisting of H, OH, SH, CN, NO₂, halo, oxo, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₁₋₆ haloalkyl, C₁₋₆ cyanoalkyl, C₁₋₆ hydroxyalkyl, C₁₋₆ alkoxy, —(C₁₋₆ alkyl)-(C₁₋₆ alkoxy), —(C₁₋₆ alkoxy)-(C₁₋₆ alkoxy), C₁₋₆ haloalkoxy, C₃₋₁₀ cycloalkyl, C₆₋₁₀ aryl, 5-10 membered heteroaryl, 4-10 membered heterocycloalkyl, amino, C₁₋₆ alkylamino, di(C₁₋₆ alkyl)amino, carbamyl, C₁₋₆ alkylcarbamyl, di(C₁₋₆ alkyl)carbamyl, carbamoyl, C₁₋₆ alkylcarbamoyl, di(C₁₋₆ alkyl)carbamoyl, C₁₋₆ alkylcarbonyl, C₁₋₆ alkoxycarbonyl, C₁₋₆ alkylcarbonylamino, C₁₋₆ alkylsulfonylamino, aminosulfonyl, C₁₋₆ alkylaminosulfonyl, di(C₁₋₆ alkyl)aminosulfonyl, aminosulfonylamino, C₁₋₆ alkylaminosulfonylamino, di(C₁₋₆ alkyl)aminosulfonylamino, aminocarbonylamino, C₁₋₆ alkylaminocarbonylamino, and di(C₁₋₆ alkyl)aminocarbonylamino. In some embodiments, each R², R², R₃, R⁴, R⁵, R⁶, and R⁷, unless specified otherwise in Table 1, is H. In some embodiments, at least one of R¹, R², R³, R⁴, and R⁵ is not H.

It is understood that the

and

shown in the substituent groups provided in Tables 1-6 represent the point of attachment between the representative Formula (e.g., Formula I, Formula II, or Formula III) and the substituent group.

TABLE 1 Substituents of Formula I

R² = CH₃

R¹ = CH₃

wherein:

  refers to the bond connecting R¹ to Formula I; and

  refers to the bond connecting R³ to Formula I

R⁴ = —OMe

R⁵ = Cl

R² = CH₃

R⁴ = —SCH₂CH₃ R³ = —OCH₃

R3 = —CH₃

R² = —CH₃

R² = —CH₃

R⁵ = —Cl

R₅ = —OCH₃

The present application further provides a compound of Formula Ia:

or a pharmaceutically acceptable salt thereof, wherein:

L is selected from the group consisting of a bond, C₁₋₄ alkylene, C₁₋₄ hydroxyalkylene, C₁₋₄ alkyleneoxy, —(C₁₋₄ alkylene)-NR^(L)—(C₁₋₄ alkylene)_(n)-, C(═O), and C(═O)NH;

R^(L) is selected from the group consisting of H and methyl;

R³ is H or C₁₋₄ alkoxy;

R⁴ is selected from the group consisting of H and piperidinyl;

Cy¹ is selected from the group consisting of phenyl, C₃₋₈ cycloalkyl, 4-6 membered heterocycloalkyl, and 4-10 membered heteroaryl;

R^(Cy) is selected from the group consisting of halo, C₁₋₄ alkyl, C₁₋₄ alkoxy, phenyl, —(C₁₋₄ alkylene)-(phenyl), C₃₋₁₀ cycloalkyl, and —(C₁₋₄ alkylene)-(C₃₋₁₀ cyloalkyl), wherein each phenyl and the phenyl group of —(C₁₋₄ alkylene)-(phenyl) is optionally substituted by 1 or 2 substituents independently selected from the group consisting of OH, halo, C₁₋₄ alkyl, and C₁₋₄ alkoxy;

n is 0 or 1; and

p is 0, 1, 2, 3, 4, or 5.

In some embodiments, L is selected from the group consisting of a bond, —CH₂—, —CH(OH)CH₂—, —CH₂CH₂NR^(L)CH₂—, —CH₂NR^(L)CH₂CH₂—, —OCH₂CH₂CH₂CH₂—, —CH₂NH—, C(═O), and C(═O)NH. In some embodiments, L is selected from the group consisting of a bond, —CH₂—, —CH(OH)CH₂—, —CH₂CH₂NHCH₂—, —CH₂N(CH₃)CH₂CH₂—, —OCH₂CH₂CH₂CH₂—, —CH₂NH—, C(═O), and C(═O)NH. In some embodiments, L is CH₂.

In some embodiments, R³ is selected from the group consisting of H and methoxy. In some embodiments, R³ is H. In some embodiments, R³ is methoxy.

In some embodiments, R⁴ is H. In some embodiments, R⁴ is piperidinyl.

In some embodiments, Cy¹ is phenyl. In some embodiments, Cy¹ is C₃₋₈ cycloalkyl. In some embodiments, Cy¹ is C₅₋₈ cycloalkyl. In some embodiments, Cy¹ is a C₃₋₈ cycloalkyl selected from the group consisting of cyclopentyl, cyclohexyl, and cycloheptyl. In some embodiments, Cy¹ is a 4-6 membered heterocycloalkyl. In some embodiments, Cy¹ is a 4-6 membered heterocycloalkyl selected from the group consisting of piperazinyl, and morpholinyl. In some embodiments, Cy¹ is piperazinyl. In some embodiments, Cy¹ is a 4-10 membered heteroaryl. In some embodiments, Cy¹ is a 4-6 membered heteroaryl. In some embodiments, Cy¹ is an 8-10 membered heteroaryl. In some embodiments, Cy¹ is a 4-10 membered heteroaryl selected from the group consisting of:

In some embodiments, R^(Cy) is selected from the group consisting of halo, C₁₋₄ alkyl, C₁₋₄ alkoxy, phenyl, —CH₂-(phenyl), C₅₋₇ cycloalkyl, and —(CH₂)-(C₅₋₇ cycloalkyl). In some embodiments, R^(Cy) is selected from the group consisting of fluoro, methyl, 2-butyl, methoxy, phenyl, —CH₂-(phenyl), cyclopentyl, cycloheptyl,

wherein each phenyl and the phenyl group of —CH₂-(phenyl) is optionally substituted by 1 or 2 substituents independently selected from the group consisting of OH, halo, C₁₋₄ alkyl, and C₁₋₄ alkoxy. In some embodiments, each phenyl and the phenyl group of —CH₂-(phenyl) is optionally substituted by 1 or 2 substituents independently selected from the group consisting of OH, fluoro, bromo, methyl, methoxy, ethoxy, and butoxy.

In some embodiments, n is 0. In some embodiments, n is 1. In some embodiments, p is 1, 2, or 3. In some embodiments, p is 3, 4, or 5. In some embodiments, p is 1 or 2. In some embodiments, p is 1. In some embodiments, p is 2. In some embodiments, p is 3. In some embodiments, p is 4. In some embodiments, p is 5.

In some embodiments, the compound of Formula Ia is selected from the group of compounds provided in Table 1A.

TABLE 1A

(1)

(29)

(31)

(40)

(6)

(15)

(28)

(14)

(43)

(3)

(9)

(33)

(7)

(12)

(19)

(41)

(42)

(32)

(13)

(27)

(8)

(36)

(20)

(21)

(4)

(59)

(16)

(26)

(22)

(34) or a pharmaceutically acceptable salt thereof. In some embodiments, the pharmaceutically acceptable salt is a hydrochloride salt.

In some embodiments, provided herein is a compound of Formula Ib:

or a pharmaceutically acceptable salt thereof, wherein, L, R⁴, and R^(Cy) are as defined above for compounds of Formula Ia.

In some embodiments, the compound is a compound of Formula Ib, or a pharmaceutically acceptable salt thereof, wherein:

L is selected from the group consisting of a bond, C₁₋₄ alkylene, C₁₋₄ hydroxyalkylene, and C(═O);

R⁴ is selected from the group consisting of H and piperidinyl; and

R^(Cy) is selected from the group consisting of C₁₋₄ alkyl, phenyl, —(C₁₋₄ alkylene)-(phenyl), C₃₋₁₀ cycloalkyl, and —(C₁₋₄ alkylene)-(C₃₋₁₀ cyloalkyl), wherein each phenyl and the phenyl group of —(C₁₋₄ alkylene)-(phenyl) is optionally substituted by 1 or 2 substituents independently selected from the group consisting of OH, halo, C₁₋₄ alkyl, and C₁₋₄ alkoxy.

In some embodiments, L is a bond. In some embodiments, L is CH₂. In some embodiments, L is C(═O). In some embodiments, L is CH(OH)CH₂.

In some embodiments, R⁴ is H. In some embodiments, R⁴ is piperidinyl. In some embodiments, R^(Cy) is selected from the group consisting of C₁₋₄ alkyl, phenyl, —CH₂-(phenyl), C₅₋₇ cycloalkyl, and —(CH₂)—(C₅₋₇ cycloalkyl), wherein each phenyl and the phenyl group of —CH₂-(phenyl) is optionally substituted by 1 or 2 substituents independently selected from the group consisting of OH, halo, C₁₋₄ alkyl, and C₁₋₄ alkoxy. In some embodiments, R^(Cy) is selected from the group consisting of methyl, 2-butyl, phenyl, —CH₂-(phenyl), cyclopentyl, cycloheptyl,

wherein each phenyl and the phenyl group of —CH₂-(phenyl) is optionally substituted by 1 or 2 substituents independently selected from the group consisting of OH, halo, C₁₋₄ alkyl, and C₁₋₄ alkoxy. In some embodiments, each phenyl and the phenyl group of —CH₂-(phenyl) is optionally substituted by 1 or 2 substituents independently selected from the group consisting of OH, fluoro, bromo, methyl, methoxy, ethoxy, and butoxy.

In some embodiments, the compound of Formula Ib is selected from the group of compounds provided in Table 1B.

TABLE 1B

(29)

(26)

(31)

(40)

(6)

(15)

(28)

(43)

(9)

(33)

(7)

(41)

(42)

(32)

(13)

(27)

(8)

(4)

(16)

(34) or a pharmaceutically acceptable salt thereof. In some embodiments, the pharmaceutically acceptable salt is a hydrochloride salt.

The present application further provides a compound of Formula Ic:

or a pharmaceutically acceptable salt thereof, wherein:

L³ is selected from the group consisting of a bond and C(O)NH;

R^(3a) is selected from the group consisting of H, C₁₋₄ alkyl, C₁₋₄ alkoxy, -phenyl-(C₁₋₄ alkylene)-R^(b), -(phenyl)-SO₂R^(b), and 5-6 membered heteroaryl, wherein the 5-6 membered heteroaryl is optionally substituted with a C₁₋₄ alkyl group;

R^(b) is selected from the group consisting of 5-6 membered heteroaryl and 5-6 membered heterocycloalkyl, wherein the 5-6 membered heterocycloalkyl is optionally substituted by a C₁₋₄ alkyl group;

L⁴ is selected from the group consisting of a bond, S, C(═O)NH, C₁₋₆ alkyleneoxy, and SO₂-Cy²-Cy³;

Cy² and Cy³ are each an independently selected 5-6 membered heterocycloalkyl group; and

R^(4a) is selected from the group consisting of H, C₁₋₄ alkyl, —(C₁₋₄ alkylene)-(C₃₋₆ cycloalkyl), 5-6 membered heterocyloalkyl, and —(C₁₋₄ alkylene)-(phenyl).

In some embodiments, L³ is a bond. In some embodiments, L³ is C(O)NH.

In some embodiments, R^(3a) is H. In some embodiments, L³ is a bond and R^(3a) is H.

In some embodiments, R^(3a) is C₁₋₄ alkoxy. In some embodiments, R^(3a) is methoxy. In some embodiments, L³ is a bond and R^(3a) is methoxy. In some embodiments, R^(3a) is -phenyl-(C₁₋₄ alkylene)-R^(b). In some embodiments, R^(3a) is -phenyl-(CH₂)—R^(b). In some embodiments, R^(3a) is -(phenyl)-SO₂R^(b). In some embodiments, R^(b) is selected from the group consisting of 5-6 membered heteroaryl and 5-6 membered heterocycloalkyl substituted by a C₁₋₄ alkyl group. In some embodiments, R^(b) is selected from the group consisting of pyridyl and 4-methylpiperidinyl. In some embodiments, R^(3a) is a 5-6 membered heteroaryl substituted with a C₁₋₄ alkyl group. In some embodiments, R^(3a) is 4-tert-butylthiazolyl.

In some embodiments, L⁴ is selected from the group consisting of a bond, S, C(═O)NH, —OCH₂CH₂OCH₂CH₂—, and SO₂-Cy²-Cy³. In some embodiments, L⁴ is a bond. In some embodiments, L⁴ is S. In some embodiments, L⁴ is C(═O)NH. In some embodiments, L⁴ is C₁₋₆ alkyleneoxy. In some embodiments, L⁴ is —OCH₂CH₂OCH₂CH₂—. In some embodiments, L⁴ is SO₂-Cy²-Cy³. In some embodiments, Cy² is a 6-membered heterocycloalkyl group comprising two ring heteroatoms which are nitrogen. In some embodiments, Cy² is piperazinyl. In some embodiments, Cy³ is a 6-membered heterocycloalkyl group comprising one ring heteroatom which is nitrogen. In some embodiments, Cy³ is piperidinyl.

In some embodiments, R^(4a) is H. In some embodiments, L⁴ is a bond and R^(4a) is H. In some embodiments, R^(4a) is C₁₋₄ alkyl. In some embodiments, R⁴ is methyl. In some embodiments, L⁴ is a bond and R^(4a) is methyl. In some embodiments, R^(4a) is —(C₁₋₄ alkylene)-(C₃₋₆ cycloalkyl). In some embodiments, R^(4a) is —(CH₂CH₂)-(C₅₋₆ cycloalkyl). In some embodiments, R^(4a) is —(CH₂CH₂)-(cyclohexyl). In some embodiments, R^(4a) is a 5-6 membered heterocyloalkyl group comprising one ring heteroatom which is nitrogen and one ring heteroatom which is oxygen. In some embodiments, R^(4a) is morpholinyl. In some embodiments, R^(4a) is —(C₁₋₄ alkylene)-(phenyl). In some embodiments, R^(4a) is —(CH₂)-(phenyl).

In some embodiments, the compound of Formula Ic is selected from the group of compounds provided in Table 1C.

TABLE 1C

(50)

(51)

(52)

(44)

(48)

(49) or a pharmaceutically acceptable salt thereof. In some embodiments, the pharmaceutically acceptable salt is a hydrochloride salt.

The present application further provides a compound of Formula II:

or a pharmaceutically acceptable salt thereof, wherein substituents R¹, R², R³, R⁴, R⁵, R⁶, and R⁷ of Formula II are as defined herein.

In some embodiments, each R¹, R², R³, R⁴, R⁵, R⁶, and R⁷, unless specified otherwise in Table 2, is selected from the group consisting of H, OH, SH, CN, NO₂, halo, oxo, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₁₋₆ haloalkyl, C₁₋₆ cyanoalkyl, C₁₋₆ hydroxyalkyl, C₁₋₆ alkoxy, —(C₁₋₆ alkyl)-(C₁₋₆ alkoxy), —(C₁₋₆ alkoxy)-(C₁₋₆ alkoxy), C₁₋₆ haloalkoxy, C₃₋₁₀ cycloalkyl, C₆₋₁₀ aryl, 5-10 membered heteroaryl, 4-10 membered heterocycloalkyl, amino, C₁₋₆ alkylamino, di(C₁₋₆ alkyl)amino, carbamyl, C₁₋₆ alkylcarbamyl, di(C₁₋₆ alkyl)carbamyl, carbamoyl, C₁₋₆ alkylcarbamoyl, di(C₁₋₆ alkyl)carbamoyl, C₁₋₆ alkylcarbonyl, C₁₋₆ alkoxycarbonyl, C₁₋₆ alkylcarbonylamino, C₁₋₆ alkylsulfonylamino, aminosulfonyl, C₁₋₆ alkylaminosulfonyl, di(C₁₋₆ alkyl)aminosulfonyl, aminosulfonylamino, C₁₋₆ alkylaminosulfonylamino, di(C₁₋₆ alkyl)aminosulfonylamino, aminocarbonylamino, C₁₋₆ alkylaminocarbonylamino, and di(C₁₋₆ alkyl)aminocarbonylamino. In some embodiments, each R¹, R², R³, R⁴, R⁵, R⁶, and R⁷, unless specified otherwise in Table 2, is H. In some embodiments, at least one of R¹, R², R³, R⁴, R⁵, R⁶, and R⁷, is not H.

TABLE 2 Substituents of Formula II

R² = OH

R² = OHC₃ R² & R⁴ = CH₃

R² = OH

wherein:

  refers to the bond connecting R² to Formula II; and

  refers to the bond connecting R³ to Formula II

R² = Cl

R² = F R¹ = CH₃ R² = isopropyl R¹ and R² = ethyl R¹ = trans-cinnamyl R² = OCH₃

R² = NO₂

R² = NO₂

R² = Cl

R² = OH

R² = F

R¹ = benzyl R² = OH R² = benzyl R³ = NO₂ R¹ = benzyl R², R⁴, R⁵ = OCH₃ R² = phenyl R² = OH R² = CH₃

R² = ethyl R¹ = phenyl R² = NO₂ R¹ = phenyl R² = Br R¹ = n-nitrophenyl R² = —OCH₂CH₃

R² = —OCH₂CH₃

R² = OH

R² = CH₃

R² = F

R² = NO₂

R² = CH₃

R² = —OCH₂CH₃

R² = —OCH₂CH₃

R² = F R¹ = —CH₂CH₂OH R² = isopropyl R¹ = —CH₂CH₂OH R² = ethyl R¹ = benzyl R² & R⁵ = —OCH₃ R¹ = —CH₂CH₂OH R² = CH₃

R², R³, and R⁵ = OCH₃ R⁶ = (═O)

R² = NO₂ R¹ = CH₃

wherein:

  refers to the bond connecting R² to Formula II; and

  refers to the bond connecting R³ to Formula II R¹ = ethyl R² = —N(ethyl)₂ R¹ = p-nitrophenyl R² = —OCH₂CH₂CH₂CH₃

R¹ = benzyl R⁵ = Cl R⁷ = OH

R⁷ = NO₂

wherein:

  refers to the bond connecting R² to Formula II; and

  refers to the bond connecting R³ to Formula II

R⁴ & R⁵ = —OCH₃

R² & R³ = —OCH₃

R³ & R⁴ = —OCH₃

R² & R⁴ = —OCH₃

R⁷ = F

R² = NO₂ R⁶ = (═O)

R⁶ = O R⁷ = OCH₃ R¹ = cyclohexyl

R⁵ = Br R⁷ = OH

R⁵ = Br R⁷ = OH

R⁷ = NO₂ R¹ = sec-butyl R² = NO₂

R² = NO₂ R¹ = cyclophenyl R² = NO₂ R¹ = cyclophenyl R² = NO₂ R¹ = ethyl R² = tert-butyl R⁶ = O

R⁶ = O

R² = —OCH₃

R² & R³ = —OCH₃

The present application further provides a compound of Formula IIa:

or a pharmaceutically acceptable salt thereof, wherein:

L¹ is selected from the group consisting of a bond, C₁₋₄ alkylene, C₂₋₄ alkenylene, C₁₋₄ hydroxyalkylene, C₁₋₄ alkyleneoxy, 4-6 membered heterocycloalkylene, and C(═O), wherein the C₁₋₄ alkyleneoxy is further optionally substituted by one OH group;

R^(3a) is selected from the group consisting of OH, C₁₋₄ alkyl, C₅₋₁₀ cycloalkyl, 8-10 membered heteroaryl, phenyl, —(C₁₋₄ alkylene)-(phenyl), and 10-13 membered heteroaryl, wherein the C₅₋₁₀ cycloalkyl, phenyl, and the phenyl group of the —(C₁₋₄ alkylene)-(phenyl) are each optionally substituted by 1, 2, or 3 substituents independently selected from the group consisting of halo, OH, NO₂, C₁₋₄ alkyl, and C₁₋₄ alkoxy, and wherein the 10-13 membered heteroaryl is optionally substituted by one halo group;

R² is selected from the group consisting of H, halo, OH, NO₂, C₁₋₄ alkyl, C₁₋₄ alkoxy, and —(C₁₋₄ alkylene)-(8-10 membered heteroaryl);

R³ is selected from the group consisting of H, halo, C₁₋₄ alkoxy, and phenoxy; or

alternatively, R² and R³ come together, in combination with the carbon atoms to which they are attached, to form a 5-6 membered heterocycloalkyl or a C₆₋₁₀ aryl;

R⁴ is selected from the group consisting of H, OH, and C₁₋₄ alkoxy;

R⁵ is selected from the group consisting of H, halo, C₁₋₄ alkoxy, and phenoxy; and

R⁶ is selected from the group consisting of H and oxo.

In some embodiments, L¹ is a bond. In some embodiments, L¹ is a C₁₋₄ alkylene. In some embodiments, L¹ is selected from the group consisting of methylene and ethylene. In some embodiments, L¹ is C₂₋₄ alkenylene. In some embodiments, L¹ is propenylene. In some embodiments, L¹ is —CH₂CH═CH—. In some embodiments, L¹ is C₁₋₄ hydroxyalkylene. In some embodiments, L¹ is —CH₂CH(OH)—. In some embodiments, L¹ is a C₁₋₄ alkyleneoxy which is further substituted by one OH group. In some embodiments, L¹ is a propoxy group which is further substituted by one OH group. In some embodiments, L¹ is —CH₂CH(OH)CH₂O—. In some embodiments, L¹ is C(═O). In some embodiments, L¹ is a 4-6 membered heterocycloalkylene. In some embodiments, L¹ is a 4-6 membered heterocycloalkylene comprising one ring heteroatom which is nitrogen. In some embodiments, L¹ is a 5-membered heterocycloalkylene comprising one ring heteroatom which is nitrogen. In some embodiments, L¹ is:

wherein:

indicates the bond between L¹ and the piperazine group of Formula II; and

indicates the bond between L¹ and R^(1a).

In some embodiments, R^(1a) is OH. In some embodiments, L¹ is a bond and R^(1a) is OH. In some embodiments, R^(1a) is a C₁₋₄ alkyl group. In some embodiments, L¹ is a bond and R^(1a) is a C₁₋₄ alkyl group. In some embodiments, R^(1a) is selected from the group consisting of methyl and ethyl. In some embodiments, R^(1a) is a C₅₋₁₀ cycloalkyl group which is optionally substituted by one C₁₋₄ alkyl group. In some embodiments, R^(1a) is a C₅₋₇ cycloalkyl group which is optionally substituted by one C₁₋₄ cycloalkyl group. In some embodiments, R^(1a) is a C₈₋₁₀ cycloalkyl group. In some embodiments, R^(1a) is selected from the group consisting of cyclopentyl, cyclohexyl, 3-methylcyclohexyl, cycloheptyl, and

In some embodiments, R^(1a) is a 8-10 membered heteroaryl group. In some embodiments, R^(1a) is a 8-10 membered heteroaryl group comprising two ring heteroatoms which are oxygen. In some embodiments, R^(1a) is a 10-membered heteroaryl group comprising two ring heteroatoms which are oxygen. In some embodiments, R^(1a) is:

In some embodiments, R^(1a) is phenyl or —(C₁₋₄ alkylene)-(phenyl), wherein the phenyl and the phenyl group of the —(C₁₋₄ alkylene)-(phenyl) are each optionally substituted by 1, 2, or 3 substituents independently selected from the group consisting of halo, OH, NO₂, C₁₋₄ alkyl, and C₁₋₄ alkoxy. In some embodiments, R^(1a) is phenyl or —(C₁₋₄ alkylene)-(phenyl), wherein the phenyl and the phenyl group of the —(C₁₋₄ alkylene)-(phenyl) are each optionally substituted by 1, 2, or 3 substituents independently selected from the group consisting of halo, OH, NO₂, C₁₋₄ alkyl, and C₁₋₄ alkoxy. In some embodiments, R^(1a) is selected from the group consisting of phenyl, —(CH₂)-(phenyl), and —(CH₂CH₂)-(phenyl), wherein the phenyl and the phenyl group of the —(CH₂)-(phenyl) and —(CH₂CH₂)-(phenyl) are each optionally substituted by 1, 2, or 3 substituents independently selected from the group consisting of halo, OH, NO₂, C₁₋₄ alkyl, and C₁₋₄ alkoxy. In some embodiments, the phenyl and the phenyl group of the —(CH₂)-(phenyl) and —(CH₂CH₂)-(phenyl) are unsubstituted. In some embodiments, the phenyl and the phenyl group of the —(CH₂)-(phenyl) and —(CH₂CH₂)-(phenyl) are substituted by 1, 2, or 3 substituents independently selected from the group consisting of halo, OH, NO₂, C₁₋₄ alkyl, and C₁₋₄ alkoxy. In some embodiments, the phenyl and the phenyl group of the —(CH₂)-(phenyl) and —(CH₂CH₂)-(phenyl) are substituted by 1, 2, or 3 substituents independently selected from the group consisting of fluoro, chloro, bromo, OH, NO₂, methyl, ethyl, 2-butyl, and methoxy.

In some embodiments, R^(1a) is a 10-13 membered heteroaryl group which is optionally substituted by one halo group. In some embodiments, R^(1a) is 10-membered heteroaryl which is substituted by one halo group. In some embodiments, R^(1a) is 10-membered heteroaryl which is substituted by one halo group and wherein the 10-membered heteroaryl comprises two ring heteroatoms which are nitrogen. In some embodiments, R^(1a) is a 13-membered heteroaryl group. In some embodiments, R^(1a) is a 13-membered heteroaryl group comprising two ring heteroatoms which are nitrogen and one ring heteroatom which is sulfur. In some embodiments, R^(1a) is selected from the group consisting of:

In some embodiments, R² is H. In some embodiments, R² is halo. In some embodiments, R² is selected from the group consisting of F and Cl. In some embodiments, R² is OH. In some embodiments, R² is C₁₋₄ alkyl. In some embodiments, R² is selected from the group consisting of methyl, ethyl, isopropyl, and tert-butyl. In some embodiments, R² is NO₂. In some embodiments, R² is C₁₋₄ alkoxy. In some embodiments, R² is selected from the group consisting of methoxy, ethoxy, and butoxy.

In some embodiments, R² is -(C₁₋₄ alkylene)-(8-10 membered heteroaryl). In some embodiments, R² is —(C₁₋₄ alkylene)-(8-10 membered heteroaryl), wherein the 8-10 membered heteroaryl group comprises one ring heteroatom which is nitrogen. In some embodiments, R² is —(CH₂)-(8-10 membered heteroaryl), wherein the 8-10 membered heteroaryl group comprises one ring heteroatom which is nitrogen. In some embodiments, R² is —(CH₂)-(10-membered heteroaryl), wherein the 10-membered heteroaryl group comprises one ring heteroatom which is nitrogen. In some embodiments, R² is:

In some embodiments, R³ is H. In some embodiments, R³ is halo. In some embodiments, R³ is selected from the group consisting of Cl and Br. In some embodiments, R³ is C₁₋₄ alkoxy or phenoxy. In some embodiments, R³ is methoxy or phenoxy.

In some embodiments, R² and R³ come together, in combination with the carbon atoms to which they are attached, to form a 5-6 membered heterocycloalkyl or a C₆₋₁₀ aryl. In some embodiments, R² and R³ come together, in combination with the carbon atoms to which they are attached, to form a 5-6 membered heterocycloalkyl. In some embodiments, R² and R³ come together, in combination with the carbon atoms to which they are attached, to form a 5-6 membered heterocycloalkyl wherein the 5-6 membered heterocycloalkyl comprises two ring atoms which are oxygen. In some embodiments, R² and R³ come together, in combination with the carbon atoms to which they are attached, to form a C₆₋₁₀ aryl. In some embodiments, R² and R³ come together, in combination with the carbon atoms to which they are attached, to form a C₇ aryl. In some embodiments, R² and R³ come together, in combination with the carbon atoms to which they are attached, to form a group selected from:

wherein:

indicates the bond formed by R² and the phenyl group; and

indicates the bond formed by R³ and the phenyl group.

In some embodiments, R⁴ is H. In some embodiments, R⁴ is OH. In some embodiments, R⁴ is methoxy.

In some embodiments, R⁵ is H. In some embodiments, R⁵ is halo. In some embodiments, R⁵ is selected from the group consisting of Cl and Br. In some embodiments, R⁵ is C₁₋₄ alkoxy or phenoxy. In some embodiments, R⁵ is methoxy or phenoxy.

In some embodiments, R⁶ is H. In some embodiments, R⁶ is oxo (i.e., ═O).

In some embodiments, the compound of Formula IIa is selected from the group of compounds provided in Table 2A.

TABLE 2A

(62)

(63)

(81)

(100)

(29)

(31)

(79)

(40)

(6)

(15)

(129)

(28)

(82)

(43)

(121)

(110)

(71)

(96)

(135)

(98)

(112)

(76)

(103)

(72)

(109)

(106)

(9)

(111)

(93)

(83)

(97)

(90)

(91)

(65)

(64)

(78)

(7)

(41)

(130)

(42)

(32)

(75)

(94)

(13)

(8)

(66)

(27)

(88)

(4)

(132)

(77)

(67)

(133)

(113)

(16)

(26)

(34)

(131)

(123)

(95) or a pharmaceutically acceptable salt thereof. In some embodiments, the pharmaceutically acceptable salt is a hydrochloride salt.

The present application further provides a compound of Formula III:

or a pharmaceutically acceptable salt thereof, wherein R¹ and R² of Formula III are as defined herein.

In some embodiments, each r¹ and R², unless specified otherwise in Table 3, is selected from the group consisting of H, OH, SH, CN, NO₂, halo, oxo, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₁₋₆ haloalkyl, C₁₋₆ cyanoalkyl, C₁₋₆ hydroxyalkyl, C₁₋₆ alkoxy, —(C₁₋₆ alkyl)-(C₁₋₆ alkoxy), —(C₁₋₆ alkoxy)-(C₁₋₆ alkoxy), C₁₋₆ haloalkoxy, C₃₋₁₀ cycloalkyl, C₆₋₁₀ aryl, 5-10 membered heteroaryl, 4-10 membered heterocycloalkyl, amino, C₁₋₆ alkylamino, di(C₁₋₆ alkyl)amino, carbamyl, C₁₋₆ alkylcarbamyl, di(C₁₋₆ alkyl)carbamyl, carbamoyl, C₁₋₆ alkylcarbamoyl, di(C₁₋₆ alkyl)carbamoyl, C₁₋₆ alkylcarbonyl, C₁₋₆ alkoxycarbonyl, C₁₋₆ alkylcarbonylamino, C₁₋₆ alkylsulfonylamino, aminosulfonyl, C₁₋₆ alkylaminosulfonyl, di(C₁₋₆ alkyl)aminosulfonyl, aminosulfonylamino, C₁₋₆ alkylaminosulfonylamino, di(C₁₋₆ alkyl)aminosulfonylamino, aminocarbonylamino, C₁₋₆ alkylaminocarbonylamino, and di(C₁₋₆ alkyl)aminocarbonylamino. In some embodiments, each R¹ and R² unless specified otherwise in Table 3, is H. In some embodiments, at least one of R¹ and R² is not H.

TABLE 3 Substituents of Formula III R¹ = R² = R² =

The present application further provides a compound of Formula IIIa:

or a pharmaceutically acceptable salt thereof, wherein:

L¹ is selected from the group consisting of a bond, NH, N(C₁₋₄ alkyl), C₂₋₄ alkynyl, NHC(═O)(C₁₋₄ thioalkyl), C₁₋₆ alkyleneoxy, 5-6 membered heterocycloalkyl, NHC(═O)C₁₋₄ alkylene, and (C₁₋₄ alkylene)-NHC(═O)—(C₁₋₄ alkylene)-, wherein the C₁₋₆ alkyleneoxy is further optionally substituted by one OH group;

R^(1a) is selected from the group consisting of H, C₁₋₄ alkyl, C₂₋₄ alkenyl, C₁₋₄ cyanoalkylene, 5-6 membered heterocycloalkyl, 5-6 membered heteroaryl, NH(C₁₋₄ alkyl), and NH(C₂₋₄ alkenyl), wherein the C₁₋₄ alkyl and C₂₋₄ alkenyl are each optionally substituted by one substituent selected from the group consisting of phenyl and hydroxyphenyl, and wherein the 5-6 membered heterocycloalkyl and 5-6 membered heteroaryl are each optionally substituted by 1 or 2 substituents independently selected from the group consisting of C₁₋₄ alkyl, NH₂ and di-halo substituted phenyl;

L² is selected from the group consisting of a bond and 5-6 membered heterocycloalkyl; and

R^(2a) is selected from the group consisting of H, C₁₋₄ hydroxyalkyl, —SO₂(C₁₋₄ alkyl).

In some embodiments, L¹ is a bond. In some embodiments, L¹ is selected from the group consisting of NH and N(C₁₋₄ alkyl). In some embodiments, L¹ is selected from the group consisting of NH and N(CH₃). In some embodiments, L¹ is C₂₋₄ alkynyl. In some embodiments, L¹ is —C≡CCH₂—. In some embodiments, L¹ is NHC(═O)(C₁₋₄ thioalkyl). In some embodiments, L¹ is L¹ is NHC(═O)CH₂S—. In some embodiments, L¹ is C₁₋₆ alkyleneoxy which is further substituted by one OH group. In some embodiments, L¹ is —CH₂CH₂OCH₂CH(OH)CH₂. In some embodiments, L¹ is a 5-6 membered heterocycloalkyl group. In some embodiments, L¹ is a 5-6 membered heterocycloalkyl group wherein the 5-6 membered heterocycloalkyl group comprises two ring heteroatoms which are nitrogen. In some embodiments, L¹ is piperazinyl. In some embodiments, L¹ is selected from the group consisting of NHC(═O)C₁₋₄ alkylene and (C₁₋₄ alkylene)-NHC(═O)—(C₁₋₄ alkylene)-. In some embodiments, L¹ is selected from the group consisting of —NHC(═O)CH₂— and —CH₂NHC(═O)CH₂—.

In some embodiments, R^(1a) is H. In some embodiments, L¹ is a bond and R^(1a) is H. In some embodiments, R^(1a) is selected from the group consisting of C₁₋₄ alkyl, C₂₋₄ alkenyl, and C₁₋₄ cyanoalkyl, wherein the C₁₋₄ alkyl and C₂₋₄ alkenyl are each optionally substituted by one substituent selected from the group consisting of phenyl and hydroxyphenyl. In some embodiments, R^(1a) is selected from the group consisting of CH₂CH₂CN,

In some embodiments, R^(1a) is selected from the group consisting of 5-6 membered heterocycloalkyl and 5-6 membered heteroaryl, wherein the 5-6 membered heterocycloalkyl and 5-6 membered heteroaryl are each optionally substituted by 1 or 2 substituents independently selected from the group consisting of C₁₋₄ alkyl, NH₂ and di-halo substituted phenyl. In some embodiments, R^(1a) is selected from the group consisting of 6-membered heterocycloalkyl and 5-membered heteroaryl, wherein the 6-membered heterocycloalkyl and 5-membered heteroaryl are each optionally substituted by 1 or 2 substituents independently selected from the group consisting of methyl, ethyl, NH₂ and di-halo substituted phenyl. In some embodiments, R^(1a) is selected from the group consisting of:

In some embodiments, R^(1a) is selected from the group consisting of NH(C₁₋₄ alkyl) and NH(C₂₋₄ alkenyl). In some embodiments, R^(1a) is NH(CH(CH₃)₂). In some embodiments, R^(1a) is NH(CH₂CH═CH₂).

In some embodiments, L² is a bond. In some embodiments, L² is a 5-6 membered heterocycloalkyl group. In some embodiments, L² is a 5-6 membered heterocycloalkyl group, wherein the 5-6 membered heterocycloalkyl group comprises two ring heteroatoms which are nitrogen. In some embodiments, L² is a 6-membered heterocycloalkyl group, wherein the 6-membered heterocycloalkyl group comprises two ring heteroatoms which are nitrogen. In some embodiments, L² is piperazinyl.

In some embodiments, R^(2a) is H. In some embodiments, L² is a bond and R^(2a) is H. In some embodiments, R^(2a) is SO₂(C₁₋₄ alkyl). In some embodiments, R^(2a) is SO₂CH₃. In some embodiments, R^(2a) is C₁₋₄ hydroxyalkyl. In some embodiments, R^(2a) is CH₂CH₂OH.

In some embodiments, the compound of Formula IIIa is selected from the group of compounds provided in Table 3A.

TABLE 3A

(144)

(136)

(143)

(149)

(138)

(145)

(137)

(139)

(140)

(146)

(142)

(147)

(141)

or a pharmaceutically acceptable salt thereof. In some embodiments, the pharmaceutically acceptable salt is a hydrochloride salt.

In some embodiments, the compound is selected from the group provided in Table 4, or a pharmaceutically acceptable salt thereof. Unless specified otherwise, each R group of Table 4 is H.

TABLE 4

Cmpd # Substituent Groups  1

 2

 3

 4

 5

 6

 7

 8

 9

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

57

58

59

60

61

In some embodiments, the compound is selected from the group provided in Table 5, or a pharmaceutically acceptable salt thereof. Unless specified otherwise, each R group of Table 5 is H.

TABLE 5

Cmpd # Substituent Groups  4

 6 R¹ = benzyl R³ = NO₂  7 R¹ = phenyl R² = NO₂  8 R¹ = p-nitrophenyl R² = —OCH₂CH₃  9

 13

 15 R¹ = p-nitrophenyl R² = —OCH₂CH₂CH₂CH₃  16

 24

 27

 28

 29

 31

 32

 34

 40 R¹ = sec-butyl R² = NO₂  41

 42 R¹ = cyclopentyl R² = NO₂  43 R¹ = cycloheptyl R² = NO₂  62

 63

 64

 65

 66

 67

 68

 69

 70 R¹ = CH₃ R² = isopropyl  71 R¹ and R² = ethyl  72 R¹ = trans-cinnamyl R² = OCH₃  74

 75

 76

 77

 78

 79 R¹ = benzyl R² = OH  81 R¹ = benzyl R², R⁴, and R⁵ = OCH₃  82 R¹ = phenyl R² = OH  83

 84

 86 R¹ = phenyl R² = Br  88

 90

 91

 93

 94

 95

 96

 97 R¹ = —CH₂CH₂OH R² = isopropyl  98 R¹ = —CH₂CH₂OH R² = ethyl  99 R¹ = benzyl R³ & R⁵ = OCH₃ 100 R¹ = —CH₂CH₂OH R² = CH₃ 101

103

104 R¹ = ethyl R² = —N(ethyl)₂ 106

107 R¹ = benzyl R⁵ ⁼ Cl R⁷ ⁼ OH 109

110

111

112

113

120

121 R¹ = cyclohexyl 123

124

129 R¹ = ethyl R² = tert-butyl R⁶ = O 130

131

132

133

134 R¹ = 135 R¹ = R² & R³ = —OCH₃

In some embodiments, the compound is selected from the group provided in Table 6, or a pharmaceutically acceptable salt thereof. Unless specified otherwise, each R group of Table 6 is H.

TABLE 6

Cmpd # Substituent Groups 136 R¹ = 137 R² = 138 R² = 139 R² = 140 R² = 141

142

143

144

145

146

147

148

149

In some embodiments, the compound is selected from the group of compounds provided in Table 1A, Table 1B, Table 1C, Table 2A, Table 3A, Table 4, Table 5, and Table 6, or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is selected from the group consisting of:

2-(3,4-dimethoxyphenyl)-N-methyl-N-(4-nitrobenzyl)ethan-1-amine;

N-methyl-N-(4-nitrobenzyl)cyclohexanamine;

3-(adamantan-1-yl(methyl)amino)propanenitrile;

1-(adamantan-2-yl)-4-(methyl sulfonyl)piperazine;

4-((4-(2-methoxyphenyl)piperazin-1-yl)methyl)phenol;

(4-benzylpiperazin-1-yl)(2,3-dihydrobenzo[b]1,4]dioxin-2-yl) methanone;

1-cinnamyl-4-(4-methoxybenzyl)piperazine;

1-(4-fluorobenzyl)-4-(o-tolyl)piperazine; and

1-benzyl-4-(bicyclo[2.2.1]heptan-2-yl)piperazine;

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is selected from the group consisting of:

2-(3,4-dimethoxyphenyl)-N-methyl-N-(4-nitrobenzyl)ethan-1-amine;N-methyl-N-(4-nitrobenzyl)cyclohexanamine;

3-(adamantan-1-yl(methyl)amino)propanenitrile hydrochloride;

1-(adamantan-2-yl)-4-(methyl sulfonyl)piperazine;

4-((4-(2-methoxyphenyl)piperazin-1-yl)methyl)phenol;

(4-benzylpiperazin-1-yl)(2,3-dihydrobenzo[b]1,4]dioxin-2-yl) methanone hydrochloride;

1-cinnamyl-4-(4-methoxybenzyl)piperazine;

1-(4-fluorobenzyl)-4-(o-tolyl)piperazine; and

1-benzyl-4-(bicyclo[2.2.1]heptan-2-yl)piperazine.

In some embodiments, the compound is selected from the group consisting of:

(2-(3,4-dimethoxyphenyl)-N-methyl-N-(4-nitrobenzyl)ethan-1-amine);

(4-((4-(2-methoxyphenyl)piperazin-1-yl)methyl)phenol); and

(3-(adamantan-1-yl(methyl)amino)propanenitrile;

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is selected from the group consisting of:

(2-(3,4-dimethoxyphenyl)-N-methyl-N-(4-nitrobenzyl)ethan-1-amine);

(4-((4-(2-methoxyphenyl)piperazin-1-yl)methyl)phenol); and

(3-(adamantan-1-yl(methyl)amino)propanenitrile;

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is selected from the group consisting of:

(2-(3,4-dimethoxyphenyl)-N-methyl-N-(4-nitrobenzyl)ethan-1-amine);

(4-((4-(2-methoxyphenyl)piperazin-1-yl)methyl)phenol); and

(3-(adamantan-1-yl(methyl)amino)propanenitrile hydrochloride).

The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds or salts which are suitable for use in contact with cells (e.g., eukaryotic cells or cyanobacteria) without excessive toxicity or other problem or complication (i.e., the contacting does not affect the viability of the cell).

Methods of Use

The present application further provides a method of increasing lipid accumulation in a cell, comprising contacting the cell with an effective amount of a compound provided herein (e.g. a compound of any one of Formulae I, Ia, Ib, Ic, II, IIa, III, and IIIa, or a pharmaceutically acceptable salt thereof). In some embodiments, the compound is a compound of Formula Ia, or a pharmaceutically acceptable salt thereof, or a compound of Formula Ib, or a pharmaceutically acceptable salt thereof, or a compound of Formula Ic, or a pharmaceutically acceptable salt thereof, or a compound of Formula IIa, or a pharmaceutically acceptable salt thereof, or a compound of Formula IIIa, or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is a compound of Formula I, or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is a compound of Formula II, or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is a compound of Formula III, or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is selected from the group of compounds provided in Table 1A, Table 1B, Table 1C, Table 2A, Table 3A, Table 4, Table 5, and Table 6, or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is selected from the group consisting of:

2-(3,4-dimethoxyphenyl)-N-methyl-N-(4-nitrobenzyl)ethan-1-amine;

N-methyl-N-(4-nitrobenzyl)cyclohexanamine;

3-(adamantan-1-yl(methyl)amino)propanenitrile;

1-(adamantan-2-yl)-4-(methyl sulfonyl)piperazine;

4-((4-(2-methoxyphenyl)piperazin-1-yl)methyl)phenol;

(4-benzylpiperazin-1-yl)(2,3-dihydrobenzo[b]1,4]dioxin-2-yl) methanone;

1-cinnamyl-4-(4-methoxybenzyl)piperazine;

1-(4-fluorobenzyl)-4-(o-tolyl)piperazine; and

1-benzyl-4-(bicyclo[2.2.1]heptan-2-yl)piperazine;

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is selected from the group consisting of:

2-(3,4-dimethoxyphenyl)-N-methyl-N-(4-nitrobenzyl)ethan-1-amine;

N-methyl-N-(4-nitrobenzyl)cyclohexanamine;

3-(adamantan-1-yl(methyl)amino)propanenitrile hydrochloride;

1-(adamantan-2-yl)-4-(methyl sulfonyl)piperazine;

4-((4-(2-methoxyphenyl)piperazin-1-yl)methyl)phenol;

(4-benzylpiperazin-1-yl)(2,3-dihydrobenzo[b]1,4]dioxin-2-yl) methanone hydrochloride;

1-cinnamyl-4-(4-methoxybenzyl)piperazine;

1-(4-fluorobenzyl)-4-(o-tolyl)piperazine; and

1-benzyl-4-(bicyclo[2.2.1]heptan-2-yl)piperazine.

In some embodiments, the compound is selected from the group consisting of:

(2-(3,4-dimethoxyphenyl)-N-methyl-N-(4-nitrobenzyl)ethan-1-amine);

(4-((4-(2-methoxyphenyl)piperazin-1-yl)methyl)phenol); and

(3-(adamantan-1-yl(methyl)amino)propanenitrile;

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is selected from the group consisting of:

(2-(3,4-dimethoxyphenyl)-N-methyl-N-(4-nitrobenzyl)ethan-1-amine);

(4-((4-(2-methoxyphenyl)piperazin-1-yl)methyl)phenol); and

(3-(adamantan-1-yl(methyl)amino)propanenitrile hydrochloride).

In some embodiments, the cell is a eukaryotic cell or a cyanobacteria. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a cyanobacteria. In some embodiments, the cell is selected from the group consisting of an algal cell, a drosophila cell, a yeast cell, or a fungus cell.

Example algal cells include, but are not limited to, Chlamydomonas reinhardtii, Chlorella sorokiniana, Tetrachlorella alterans, C. protothecoides, C. vulgaris, Nannochloropsis sp., P. tricornutum, N. salina, Nannochloris sp., N. oculata, Clostridium acetobutylicum, Botryococcus braunii, Dunaliella tertiolecta, Gracilaria, Chlorella sp., Pleurochrysis carterae, Sargassum, Ankistrodesmus,

Example yeast cells include, but are not limited to, Yarrowia hpolytica, Saccharomyces cerevisiae, Brettanomyces bruxellensis, Candida stellata, Schizosaccharomyces pombe, Torulaspora delbrueckii, and Zygosaccharomyces bailii.

Example fungus cells include, but are not limited to, Gliocladium roseum, Cunninghamella japonica, and Tricoderma reesei.

In some embodiments, the cell is an algal cell. In some embodiments, the cell is selected from the group consisting of:

Chlamydomonas reinhardtii;

Chlorella sorokiniana;

Tetrachlorella alterans;

C. protothecoides;

C. vulgaris; and

Nannochloropsis sp.

In some embodiments, the cell is an algal cell. In some embodiments, the cell is selected from the group consisting of:

Chlamydomonas reinhardtii CC 125;

Chlorella sorokiniana UTEX 1230;

Tetrachlorella alterans UTEX 2453;

C. protothecoides;

C. vulgaris UTEX 395; and

Nannochloropsis sp.

In some embodiments, the increasing lipid accumulation comprises increasing fatty acid accumulation. In some embodiments, the increasing fatty acid accumulation comprises increasing C₁₀-₃₀ fatty acid accumulation. In some embodiments, the increasing fatty acid accumulation comprises increasing C₁₀-₂₀ fatty acid accumulation. In some embodiments, the increasing fatty acid accumulation comprises increasing C₁₆-₁₈ fatty acid accumulation. In some embodiments, increasing lipid accumulation comprises increasing polyunsaturated fatty acid accumulation. In some embodiments, the increasing polyunsaturated fatty acid accumulation comprises increasing C₁₀-₃₀ polyunsaturated fatty acid accumulation. In some embodiments, the increasing polyunsaturated fatty acid accumulation comprises increasing C₁₀-₂₀ polyunsaturated fatty acid accumulation. In some embodiments, the increasing polyunsaturated fatty acid accumulation comprises increasing C₁₆-₁₈ polyunsaturated fatty acid accumulation.

In some embodiments, the increasing lipid accumulation comprises increasing triacylglycerol accumulation. In some embodiments, the increasing triacylglycerol accumulation comprises increasing C₃₀-₆₀ triacylglycerol accumulation. In some embodiments, the increasing triacylglycerol accumulation comprises increasing C₄₀-₆₀ triacylglycerol accumulation. In some embodiments, the triacylglycerol comprises fatty acids of C₁₀-₂₀ in chain length. In some embodiments, the triacylglycerol comprises fatty acids of C₁₅-₂₀ in chain length. In some embodiments, the triacylglycerol comprises fatty acids of 16 carbons in chain length. In some embodiments, the triacylglycerol comprises fatty acids of 18 carbons in chain length. In some embodiments, the triacylglycerol comprises fatty acids of 16 carbons and 18 carbons in chain length.

In some embodiments, the increasing lipid accumulation comprises increasing fatty acid accumulation and increasing triacylglycerol accumulation. In some embodiments, the increasing lipid accumulation comprises increasing polyunsaturated fatty acid accumulation and increasing triacylglycerol accumulation.

In some embodiments, the method further comprises increasing carbohydrate accumulation in the cell. For example, the carbohydrate accumulation may be increased by about 1.1 to about 5 fold, from about 1.25 to about 5 fold, from about 1.5 to about 5 fold, from about 1.75 to about 5 fold, from about 2 to about 5 fold, from about 2.25 to about 5 fold, from about 2.5 to about 5 fold, from about 2.75 to about 5 fold, from about 3 to about 5 fold, from about 3.25 to about 5 fold, from about 3.5 to about 5 fold, from about 3.75 to about 5 fold, from about 4 to about 5 fold, from about 4.25 to about 5 fold, from about 4.5 to about 5 fold, or from about 4.75 to about 5 fold. In some embodiments, the method further comprises increasing carbohydrate accumulation in the cell by about 1.25 to about 3.25 fold. In some embodiments, the method further comprises increasing carbohydrate accumulation in the cell by about 1.25 fold. In some embodiments, the method further comprises increasing carbohydrate accumulation in the cell by about 2 fold. In some embodiments, the method further comprises increasing carbohydrate accumulation in the cell by about 3.2 fold.

In some embodiments, the method further comprises increasing carbohydrate production in the cell. For example, the carbohydrate production may be increased by about 1.1 to about 5 fold, from about 1.25 to about 5 fold, from about 1.5 to about 5 fold, from about 1.75 to about 5 fold, from about 2 to about 5 fold, from about 2.25 to about 5 fold, from about 2.5 to about 5 fold, from about 2.75 to about 5 fold, from about 3 to about 5 fold, from about 3.25 to about 5 fold, from about 3.5 to about 5 fold, from about 3.75 to about 5 fold, from about 4 to about 5 fold, from about 4.25 to about 5 fold, from about 4.5 to about 5 fold, or from about 4.75 to about 5 fold. In some embodiments, the method further comprises increasing carbohydrate production in the cell by about 1.25 to about 3.25 fold. In some embodiments, the method further comprises increasing carbohydrate production in the cell by about 1.25 fold. In some embodiments, the method further comprises increasing carbohydrate production in the cell by about 2 fold. In some embodiments, the method further comprises increasing carbohydrate production in the cell by about 3.2 fold.

In some embodiments, the method further comprises increasing carbohydrate accumulation in the cell and increasing carbohydrate production in the cell.

In some embodiments, the lipid accumulation is increased by greater than about 1.5 fold as compared to a cell that has not been contacted by the compound, for example, greater than about 1.5 fold, greater than about 2 fold, greater than about 2.5 fold, greater than about 3 fold, greater than about 3.5 fold, greater than about 4 fold, greater than about 4.5 fold, or greater than about 5 fold as compared to a cell that has not been contacted by the compound. In some embodiments, the lipid accumulation is increased by greater than about 2-fold as compared to a cell that has not been contacted by the compound. In some embodiments, the lipid accumulation is increased by greater than about 2.5-fold as compared to a cell that has not been contacted by the compound.

In some embodiments, the contacting the cell with the compound does not reduce the rate of cellular growth compared to a cell that has not been contacted by the compound.

In some embodiments, contacting the cell with the compound does not reduce the rate of photosynthesis of the cell compared to a cell that has not been contacted by the compound. For example, the rate of photosynthesis of the cell is not reduced if the amount of Chlorophyll A and B, carotenoid, and RuBisCO (Ribulose-1,5-bisphosphate carboxylase/oxygenase) in a cell contacted by a compound provided herein are about equivalent to a cell that has not been contacted by the compound. In some embodiments, the amount of Chlorophyll A and B, carotenoid, and RuBisCO (Ribulose-1,5-bisphosphate carboxylase/oxygenase) in a cell that has been contacted by a compound provided herein are about equivalent to a cell that has not been contacted by the compound.

In some embodiments, the contacting does not adversely affect the viability of the cell. For example, the viability of the cell may include, but is not limited to, cellular growth, total cellular protein levels, the level of Chlorophyll A in a cell, the level of Chlorophyll B in a cell, the level of carotenoid in a cell, the level of RuBisCO (Ribulose-1,5-bisphosphate carboxylase/oxygenase) in a cell, the level of carbohydrates in a cell, the level of nitrogen in a cell, the level of sulfur in a cell, and the level of metal content in a cell, or any combination thereof.

In some embodiments, the lipid accumulation is increased without exposing the cell to environmental stress. As used herein, the term “environmental stress” refers to a stress upon the cell that may result in a decrease or loss of cell viability. In some embodiments, the environmental stress comprises nutrient deprivation. In some embodiments, the environmental stress comprises depriving the cell of nitrogen, depriving the cell of sulfur, depriving the cell of metal, or any combination thereof.

The present application further provides a method of increasing lipid production in a cell, comprising contacting the cell with an effective amount of a compound provided herein (e.g. a compound of any one of Formulae I, Ia, Ib, Ic, II, IIa, III, and IIIa, or a pharmaceutically acceptable salt thereof). In some embodiments, the compound is a compound of Formula Ia, or a pharmaceutically acceptable salt thereof, or a compound of Formula lb, or a pharmaceutically acceptable salt thereof, or a compound of Formula Ic, or a pharmaceutically acceptable salt thereof, or a compound of Formula IIa, or a pharmaceutically acceptable salt thereof, or a compound of Formula IIIa, or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is a compound of Formula I, or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is a compound of Formula II, or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is a compound of Formula III, or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is selected from the group of compounds provided in Table 1A, Table 1B, Table 1C, Table 2A, Table 3A, Table 4, Table 5, and Table 6, or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is selected from the group consisting of:

2-(3,4-dimethoxyphenyl)-N-methyl-N-(4-nitrobenzyl)ethan-1-amine;

N-methyl-N-(4-nitrobenzyl)cyclohexanamine;

3-(adamantan-1-yl(methyl)amino)propanenitrile;

1-(adamantan-2-yl)-4-(methyl sulfonyl)piperazine;

4-((4-(2-methoxyphenyl)piperazin-1-yl)methyl)phenol;

(4-benzylpiperazin-1-yl)(2,3-dihydrobenzo[b]1,4]dioxin-2-yl) methanone;

1-cinnamyl-4-(4-methoxybenzyl)piperazine;

1-(4-fluorobenzyl)-4-(o-tolyl)piperazine; and

1-benzyl-4-(bicyclo[2.2.1]heptan-2-yl)piperazine;

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is selected from the group consisting of:

2-(3,4-dimethoxyphenyl)-N-methyl-N-(4-nitrobenzyl)ethan-1-amine;

N-methyl-N-(4-nitrobenzyl)cyclohexanamine;

3-(adamantan-1-yl(methyl)amino)propanenitrile hydrochloride;

1-(adamantan-2-yl)-4-(methyl sulfonyl)piperazine;

4-((4-(2-methoxyphenyl)piperazin-1-yl)methyl)phenol;

(4-benzylpiperazin-1-yl)(2,3-dihydrobenzo[b]1,4]dioxin-2-yl) methanone hydrochloride;

1-cinnamyl-4-(4-methoxybenzyl)piperazine;

1-(4-fluorobenzyl)-4-(o-tolyl)piperazine; and

1-benzyl-4-(bicyclo[2.2.1]heptan-2-yl)piperazine.

In some embodiments, the compound is selected from the group consisting of:

(2-(3,4-dimethoxyphenyl)-N-methyl-N-(4-nitrobenzyl)ethan-1-amine);

(4-((4-(2-methoxyphenyl)piperazin-1-yl)methyl)phenol); and

(3-(adamantan-1-yl(methyl)amino)propanenitrile;

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is selected from the group consisting of:

(2-(3,4-dimethoxyphenyl)-N-methyl-N-(4-nitrobenzyl)ethan-1-amine);

(4-((4-(2-methoxyphenyl)piperazin-1-yl)methyl)phenol); and

(3-(adamantan-1-yl(methyl)amino)propanenitrile hydrochloride).

In some embodiments, the compound is selected from the group consisting of:

(2-(3,4-dimethoxyphenyl)-N-methyl-N-(4-nitrobenzyl)ethan-1-amine);

(4-((4-(2-methoxyphenyl)piperazin-1-yl)methyl)phenol); and

(3-(adamantan-1-yl(methyl)amino)propanenitrile;

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is selected from the group consisting of:

(2-(3,4-dimethoxyphenyl)-N-methyl-N-(4-nitrobenzyl)ethan-1-amine);

(4-((4-(2-methoxyphenyl)piperazin-1-yl)methyl)phenol); and

(3-(adamantan-1-yl(methyl)amino)propanenitrile hydrochloride).

In some embodiments, the cell is a eukaryotic cell or a cyanobacteria. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a cyanobacteria. In some embodiments, the cell is selected from the group consisting of an algal cell, a drosophila cell, a yeast cell, or a fungus cell (e.g., an algal cell, a drosophila cell, a yeast cell, or a fungus cell provided herein).

In some embodiments, the cell is an algal cell. In some embodiments, the cell is selected from the group consisting of:

Chlamydomonas reinhardtii;

Chlorella sorokiniana;

Tetrachlorella alterans;

C. protothecoides;

C. vulgaris; and

Nannochloropsis sp.

In some embodiments, the cell is an algal cell. In some embodiments, the cell is selected from the group consisting of:

Chlamydomonas reinhardtii CC 125;

Chlorella sorokiniana UTEX 1230;

Tetrachlorella alterans UTEX 2453;

C. protothecoides;

C. vulgaris UTEX 395; and

Nannochloropsis sp.

In some embodiments, the increasing lipid production comprises increasing fatty acid production. In some embodiments, the increasing fatty acid production comprises increasing C₁₀-₃₀ fatty acid production. In some embodiments, the increasing fatty acid production comprises increasing C₁₀-₂₀ fatty acid production. In some embodiments, the increasing fatty acid production comprises increasing C₁₆-₁₈ fatty acid production. In some embodiments, increasing lipid production comprises increasing polyunsaturated fatty acid production. In some embodiments, the increasing polyunsaturated fatty acid production comprises increasing C₁₀-₃₀ polyunsaturated fatty acid production. In some embodiments, the increasing polyunsaturated fatty acid production comprises increasing C₁₀-₂₀ polyunsaturated fatty acid production. In some embodiments, the increasing polyunsaturated fatty acid production comprises increasing C₁₆-₁₈ polyunsaturated fatty acid production.

In some embodiments, the increasing lipid production comprises increasing triacylglycerol production. In some embodiments, the increasing triacylglycerol production comprises increasing C₃₀-₆₀ triacylglycerol production. In some embodiments, the increasing triacylglycerol production comprises increasing C₄₀-₆₀ triacylglycerol production. In some embodiments, the triacylglycerol comprises fatty acids of C₁₀-₂₀ in chain length. In some embodiments, the triacylglycerol comprises fatty acids of C₁₅-₂₀ in chain length. In some embodiments, the triacylglycerol comprises fatty acids of 16 carbons in chain length. In some embodiments, the triacylglycerol comprises fatty acids of 18 carbons in chain length. In some embodiments, the triacylglycerol comprises fatty acids of 16 carbons and 18 carbons in chain length.

In some embodiments, the increasing lipid production comprises increasing fatty acid production and increasing triacylglycerol production. In some embodiments, the increasing lipid production comprises increasing polyunsaturated fatty acid production and increasing triacylglycerol production.

In some embodiments, the method further comprises increasing carbohydrate accumulation in the cell. For example, the carbohydrate accumulation may be increased by about 1.1 to about 5 fold, from about 1.25 to about 5 fold, from about 1.5 to about 5 fold, from about 1.75 to about 5 fold, from about 2 to about 5 fold, from about 2.25 to about 5 fold, from about 2.5 to about 5 fold, from about 2.75 to about 5 fold, from about 3 to about 5 fold, from about 3.25 to about 5 fold, from about 3.5 to about 5 fold, from about 3.75 to about 5 fold, from about 4 to about 5 fold, from about 4.25 to about 5 fold, from about 4.5 to about 5 fold, or from about 4.75 to about 5 fold. In some embodiments, the method further comprises increasing carbohydrate accumulation in the cell by about 1.25 to about 3.25 fold. In some embodiments, the method further comprises increasing carbohydrate accumulation in the cell by about 1.25 fold. In some embodiments, the method further comprises increasing carbohydrate accumulation in the cell by about 2 fold. In some embodiments, the method further comprises increasing carbohydrate accumulation in the cell by about 3.2 fold.

In some embodiments, the method further comprises increasing carbohydrate production in the cell. For example, the carbohydrate production may be increased by about 1.1 to about 5 fold, from about 1.25 to about 5 fold, from about 1.5 to about 5 fold, from about 1.75 to about 5 fold, from about 2 to about 5 fold, from about 2.25 to about 5 fold, from about 2.5 to about 5 fold, from about 2.75 to about 5 fold, from about 3 to about 5 fold, from about 3.25 to about 5 fold, from about 3.5 to about 5 fold, from about 3.75 to about 5 fold, from about 4 to about 5 fold, from about 4.25 to about 5 fold, from about 4.5 to about 5 fold, or from about 4.75 to about 5 fold. In some embodiments, the method further comprises increasing carbohydrate production in the cell by about 1.25 to about 3.25 fold. In some embodiments, the method further comprises increasing carbohydrate production in the cell by about 1.25 fold. In some embodiments, the method further comprises increasing carbohydrate production in the cell by about 2 fold. In some embodiments, the method further comprises increasing carbohydrate production in the cell by about 3.2 fold.

In some embodiments, the method further comprises increasing carbohydrate accumulation in the cell and increasing carbohydrate production in the cell.

In some embodiments, the lipid production is increased by greater than about 1.5 fold as compared to a cell that has not been contacted by the compound, for example, greater than about 1.5 fold, greater than about 2 fold, greater than about 2.5 fold, greater than about 3 fold, greater than about 3.5 fold, greater than about 4 fold, greater than about 4.5 fold, or greater than about 5 fold as compared to a cell that has not been contacted by the compound. In some embodiments, the lipid production is increased by greater than about 2-fold as compared to a cell that has not been contacted by the compound. In some embodiments, the lipid production is increased by greater than about 2.5-fold as compared to a cell that has not been contacted by the compound.

In some embodiments, the contacting the cell with the compound does not reduce the rate of cellular growth compared to a cell that has not been contacted by the compound.

In some embodiments, contacting the cell with the compound does not reduce the rate of photosynthesis of the cell compared to a cell that has not been contacted by the compound. For example, the rate of photosynthesis of the cell is not reduced if the amount of Chlorophyll A and B, carotenoid, and RuBisCO (Ribulose-1,5-bisphosphate carboxylase/oxygenase) in a cell contacted by a compound provided herein are about equivalent to a cell that has not been contacted by the compound. In some embodiments, the amount of Chlorophyll A and B, carotenoid, and RuBisCO (Ribulose-1,5-bisphosphate carboxylase/oxygenase) in a cell that has been contacted by a compound provided herein are about equivalent to a cell that has not been contacted by the compound.

In some embodiments, the contacting does not adversely affect the viability of the cell. For example, the viability of the cell may include, but is not limited to, cellular growth, total cellular protein levels, the level of Chlorophyll A in a cell, the level of Chlorophyll B in a cell, the level of carotenoid in a cell, the level of RuBisCO (Ribulose-1,5-bisphosphate carboxylase/oxygenase) in a cell, the level of carbohydrates in a cell, the level of nitrogen in a cell, the level of sulfur in a cell, and the level of metal content in a cell, or any combination thereof.

In some embodiments, the lipid production is increased without exposing the cell to environmental stress. In some embodiments, the environmental stress comprises nutrient deprivation. In some embodiments, the environmental stress comprises depriving the cell of nitrogen, depriving the cell of sulfur, depriving the cell of metal, or any combination thereof.

The present application further provides a method of producing biofuel, the method comprising:

a) contacting a cell with an effective amount of a compound provided herein (e.g., a compound of any one of Formulae I, Ia, Ib, Ic, II, IIa, III, and IIIa, or a pharmaceutically acceptable salt thereof);

b) waiting a time sufficient to allow for increased lipid accumulation; and

c) processing the cells into biofuel.

In some embodiments, the compound is a compound of Formula Ia, or a pharmaceutically acceptable salt thereof, or a compound of Formula Ib, or a pharmaceutically acceptable salt thereof, or a compound of Formula Ic, or a pharmaceutically acceptable salt thereof, or a compound of Formula IIa, or a pharmaceutically acceptable salt thereof, or a compound of Formula IIIa, or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is a compound of Formula I, or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is a compound of Formula II, or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is a compound of Formula III, or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is selected from the group of compounds provided in Table 1A, Table 1B, Table 1C, Table 2A, Table 3A, Table 4, Table 5, and Table 6, or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is selected from the group consisting of:

2-(3,4-dimethoxyphenyl)-N-methyl-N-(4-nitrobenzyl)ethan-1-amine;

N-methyl-N-(4-nitrobenzyl)cyclohexanamine;

3-(adamantan-1-yl(methyl)amino)propanenitrile;

1-(adamantan-2-yl)-4-(methyl sulfonyl)piperazine;

4-((4-(2-methoxyphenyl)piperazin-1-yl)methyl)phenol;

(4-benzylpiperazin-1-yl)(2,3-dihydrobenzo[b]1,4]dioxin-2-yl) methanone;

1-cinnamyl-4-(4-methoxybenzyl)piperazine;

1-(4-fluorobenzyl)-4-(o-tolyl)piperazine; and

1-benzyl-4-(bicyclo[2.2.1]heptan-2-yl)piperazine;

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is selected from the group consisting of:

2-(3,4-dimethoxyphenyl)-N-methyl-N-(4-nitrobenzyl)ethan-1-amine;

N-methyl-N-(4-nitrobenzyl)cyclohexanamine;

3-(adamantan-1-yl(methyl)amino)propanenitrile hydrochloride;

1-(adamantan-2-yl)-4-(methyl sulfonyl)piperazine;

4-((4-(2-methoxyphenyl)piperazin-1-yl)methyl)phenol;

(4-benzylpiperazin-1-yl)(2,3-dihydrobenzo[b]1,4] dioxin-2-yl) methanone hydrochloride;

1-cinnamyl-4-(4-methoxybenzyl)piperazine;

1-(4-fluorobenzyl)-4-(o-tolyl)piperazine; and

1-benzyl-4-(bicyclo[2.2.1]heptan-2-yl)piperazine.

In some embodiments, the compound is selected from the group consisting of:

(2-(3,4-dimethoxyphenyl)-N-methyl-N-(4-nitrobenzyl)ethan-1-amine);

(4-((4-(2-methoxyphenyl)piperazin-1-yl)methyl)phenol); and

(3-(adamantan-1-yl(methyl)amino)propanenitrile;

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is selected from the group consisting of:

(2-(3,4-dimethoxyphenyl)-N-methyl-N-(4-nitrobenzyl)ethan-1-amine);

(4-((4-(2-methoxyphenyl)piperazin-1-yl)methyl)phenol); and

(3-(adamantan-1-yl(methyl)amino)propanenitrile hydrochloride).

In some embodiments, the compound is selected from the group consisting of:

(2-(3,4-dimethoxyphenyl)-N-methyl-N-(4-nitrobenzyl)ethan-1-amine);

(4-((4-(2-methoxyphenyl)piperazin-1-yl)methyl)phenol); and

(3-(adamantan-1-yl(methyl)amino)propanenitrile;

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is selected from the group consisting of:

(2-(3,4-dimethoxyphenyl)-N-methyl-N-(4-nitrobenzyl)ethan-1-amine);

(4-((4-(2-methoxyphenyl)piperazin-1-yl)methyl)phenol); and

(3-(adamantan-1-yl(methyl)amino)propanenitrile;

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is selected from the group consisting of:

(2-(3,4-dimethoxyphenyl)-N-methyl-N-(4-nitrobenzyl)ethan-1-amine);

(4-((4-(2-methoxyphenyl)piperazin-1-yl)methyl)phenol); and

(3-(adamantan-1-yl(methyl)amino)propanenitrile hydrochloride).

In some embodiments, the cell is a eukaryotic cell or a cyanobacteria. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a cyanobacteria. In some embodiments, the cell is selected from the group consisting of an algal cell, a drosophila cell, a yeast cell, or a fungus cell (e.g., an algal cell, a drosophila cell, a yeast cell, or a fungus cell provided herein).

In some embodiments, the cell is an algal cell. In some embodiments, the cell is selected from the group consisting of:

Chlamydomonas reinhardtii;

Chlorella sorokiniana;

Tetrachlorella alterans;

C. protothecoides;

C. vulgaris; and

Nannochloropsis sp.

In some embodiments, the cell is an algal cell. In some embodiments, the cell is selected from the group consisting of:

Chlamydomonas reinhardtii CC125;

Chlorella sorokiniana UTEX 1230;

Tetrachlorella alterans UTEX 2453;

C. protothecoides;

C. vulgaris UTEX 395; and

Nannochloropsis sp.

In some embodiments, the time sufficient to allow for increased lipid accumulation is from about 1 hour to about 5 days, for example, from about 1 hour to about 5 days, from about 1 hour to about 4 days, from about 1 hour to about 3 days, from about 1 hour to about 2 days, from about 1 hour to about 1 day, from about 1 hour to about 18 hours, from about 1 hour to about 12 hours, from about 1 hour to about 6 hours, from about 1 hour to about 2 hours, from about 2 hours to about 4 days, from about 2 hours to about 3 days, from about 2 hours to about 2 days, from about 2 hours to about 1 day, from about 2 hours to about 18 hours, from about 2 hours to about 12 hours, from about 2 hours to about 6 hours, from about 6 hours to about 4 days, from about 6 hours to about 3 days, from about 6 hours to about 2 days, from about 6 hours to about 1 day, from about 6 hours to about 18 hours, from about 6 hours to about 12 hours, from about 12 hours to about 4 days, from about 12 hours to about 3 days, from about 12 hours to about 2 days, from about 12 hours to about 1 day, from about 12 hours to about 18 hours, from about 18 hours to about 4 days, from about 18 hours to about 3 days, from about 18 hours to about 2 days, from about 18 hours to about 1 day, from about 1 day to about 4 days, from about 1 day to about 3 days, from about 1 day to about 2 days, from about 2 days to about 4 days, from about 2 days to about 3 days, or from about 3 days to about 4 days. In some embodiments, the time sufficient to allow for increased lipid accumulation is about 1 day. In some embodiments, the time sufficient to allow for increased lipid accumulation is about 2 days. In some embodiments, the time sufficient to allow for increased lipid accumulation is about 3 days.

Methods of processing cells into biofuel are known in the art and would be readily understood by one of ordinary skill. For example, the cells may be processed into biofuels selected from the group consisting of biodiesel, bioalcohol (e.g., bioethanol, biopropanol, biobutanol), and cellulosic ethanol. Examples of methods for processing cells into biofuel may be found, e.g., in Pandey et al., Biofuels from Algae, 1st Ed., Elsevier B.V. (2014), the disclosure of which is incorporated herein in its entirety.

The present application further provides a nutraceutical comprising one or more lipids produced according to a method provided herein. In some embodiments, the one or more lipids is produced in a cell provided herein, wherein the cell has been contacted with a compound provided herein (e.g., a compound of any one of Formulae I, Ia, Ib, Ic, II, IIa, III, and IIIa, or a pharmaceutically acceptable salt thereof). In some embodiments, the lipid is docosahexaenoic acid (DHA). In some embodiments, the nutraceutical is formulated for oral administration, for example, as a pill, a tablet, a syrup, a drink, and the like.

Definitions

For the terms “for example” and “such as” and grammatical equivalences thereof, the phrase “and without limitation” is understood to follow unless explicitly stated otherwise. As used herein, the term “about” is meant to account for variations due to experimental error. All measurements reported herein are understood to be modified by the term “about”, whether or not the term is explicitly used, unless explicitly stated otherwise. As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

As used herein, the phrase “optionally substituted” means unsubstituted or substituted. As used herein, the term “substituted” means that a hydrogen atom is removed and replaced by a substituent. It is to be understood that substitution at a given atom is limited by valency.

Throughout the definitions, the term “C_(n-m)” indicates a range which includes the endpoints, wherein n and m are integers and indicate the number of carbons. Examples include C_(1-4,) C₁₋₆, and the like.

As used herein, the term “C_(n-m) alkyl”, employed alone or in combination with other terms, refers to a saturated hydrocarbon group that may be straight-chain or branched, having n to m carbons. Examples of alkyl moieties include, but are not limited to, chemical groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl; higher homologs such as 2-methyl-1-butyl, n-pentyl, 3-pentyl, n-hexyl, 1,2,2-trimethylpropyl, and the like. In some embodiments, the alkyl group contains from 1 to 6 carbon atoms, from 1 to 4 carbon atoms, from 1 to 3 carbon atoms, or 1 to 2 carbon atoms.

As used herein, “C_(n-m) alkenyl” refers to an alkyl group having one or more double carbon-carbon bonds and having n to m carbons. Example alkenyl groups include, but are not limited to, ethenyl, n-propenyl, isopropenyl, n-butenyl, sec-butenyl, and the like. In some embodiments, the alkenyl moiety contains 2 to 6, 2 to 4, or 2 to 3 carbon atoms.

As used herein, “C_(n-m) alkynyl” refers to an alkyl group having one or more triple carbon-carbon bonds and having n to m carbons. Example alkynyl groups include, but are not limited to, ethynyl, propyn-1-yl, propyn-2-yl, and the like. In some embodiments, the alkynyl moiety contains 2 to 6, 2 to 4, or 2 to 3 carbon atoms.

As used herein, the term “C_(n-m) alkylene”, employed alone or in combination with other terms, refers to a divalent alkyl linking group having n to m carbons. Examples of alkylene groups include, but are not limited to, ethan-1,2-diyl, propan-1,3-diyl, propan-1,2-diyl, butan-1,4-diyl, butan-1,3-diyl, butan-1,2-diyl, 2-methyl-propan-1,3-diyl, and the like. In some embodiments, the alkylene moiety contains 2 to 6, 2 to 4, 2 to 3, 1 to 6, 1 to 4, or 1 to 2 carbon atoms.

As used herein, the term “Cn-m alkenylene”, employed alone or in combination with other terms, refers to a divalent alkenyl linking group having n to m carbons. Examples of alkenylene groups include, but are not limited to, —CH═CH—, —CH═CHCH₂—, —CH₂CH₂CH═CHCH₂, and the like. In some embodiments, the alkylene moiety contains 2 to 6, 2 to 4, or 2 to 3 carbon atoms.

As used herein, the term “C_(n-m) alkoxy”, employed alone or in combination with other terms, refers to a group of formulae “-O-alkyl”, or “—(O-alkylene)_(p)-”, or “-(alkylene-O)_(p)—, wherein the alkyl or alkylene group has n to m carbons and p is an integer from 1 to 6. Example alkoxy groups include methoxy, ethoxy, propoxy (e.g., n-propoxy and isopropoxy), tert-butoxy, —(CH₂OCH₂OCH₂)—, —(CH₂CH₂OCH₂CH₂O)—, —(OCH₂)—, —(OCH₂OCH₂CH₂)—, —(CH₂CH₂OCH₂CH₂O)—, —(CH₂CH₂OCH₂CH₂OCH₂CH₂)— and the like. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms. In some embodiments, p is 1. In some embodiments, p is 2. In some embodiments, p is 3. In some embodiments, p is 4. In some embodiments, p is 5. In some embodiments, p is 6.

As used herein, the term “C_(n-m) alkylamino” refers to a group of formula —NH(alkyl), wherein the alkyl group has n to m carbon atoms. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

As used herein, the term “C_(n-m) alkoxycarbonyl” refers to a group of formula —C(O)O-alkyl, wherein the alkyl group has n to m carbon atoms. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

As used herein, the term “C_(n-m) alkylcarbonyl” refers to a group of formula —C(O)-alkyl, wherein the alkyl group has n to m carbon atoms. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

As used herein, the term “Cn-m alkylcarbonylamino” refers to a group of formula —NHC(O)-alkyl, wherein the alkyl group has n to m carbon atoms. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

As used herein, the term “C_(n-m) alkylsulfonylamino” refers to a group of formula —NHS(O)₂-alkyl, wherein the alkyl group has n to m carbon atoms. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

As used herein, the term “aminosulfonyl” refers to a group of formula —S(O)₂NH₂.

As used herein, the term “C_(n-m) alkylaminosulfonyl” refers to a group of formula —S(O)₂NH(alkyl), wherein the alkyl group has n to m carbon atoms. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

As used herein, the term “di(C_(n-m) alkyl)aminosulfonyl” refers to a group of formula —S(O)₂N(alkyl)₂, wherein each alkyl group independently has n to m carbon atoms. In some embodiments, each alkyl group has, independently, 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

As used herein, the term “aminosulfonylamino” refers to a group of formula —NHS(O)₂NH₂.

As used herein, the term “C_(n-m) alkylaminosulfonylamino” refers to a group of formula —NHS(O)₂NH(alkyl), wherein the alkyl group has n to m carbon atoms. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

As used herein, the term “di(C_(n-m) alkyl)aminosulfonylamino” refers to a group of formula —NHS(O)₂N(alkyl)₂, wherein each alkyl group independently has n to m carbon atoms. In some embodiments, each alkyl group has, independently, 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

As used herein, the term “aminocarbonylamino”, employed alone or in combination with other terms, refers to a group of formula —NHC(O)NH₂.

As used herein, the term “C_(n-m) alkylaminocarbonylamino” refers to a group of formula —NHC(O)NH(alkyl), wherein the alkyl group has n to m carbon atoms. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

As used herein, the term “di(C_(n-m) alkyl)aminocarbonylamino” refers to a group of formula —NHC(O)N(alkyl)₂, wherein each alkyl group independently has n to m carbon atoms. In some embodiments, each alkyl group has, independently, 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

As used herein, the term “C_(n-m) alkylcarbamyl” refers to a group of formula —C(O)—NH(alkyl), wherein the alkyl group has n to m carbon atoms. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

As used herein, the term “thio” refers to a group of formula —SH.

As used herein, the term “C_(n-m) alkylsulfinyl” refers to a group of formula —S(O)-alkyl, wherein the alkyl group has n to m carbon atoms. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

As used herein, the term “C_(n-m) alkylsulfonyl” refers to a group of formula —S(O)₂-alkyl, wherein the alkyl group has n to m carbon atoms. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

As used herein, the term “amino” refers to a group of formula —NH₂.

As used herein, the term “aryl,” employed alone or in combination with other terms, refers to an aromatic hydrocarbon group, which may be monocyclic or polycyclic (e.g., having 2, 3 or 4 fused rings). The term “C_(n-m) aryl” refers to an aryl group having from n to m ring carbon atoms. Aryl groups include, e.g., phenyl, naphthyl, anthracenyl, phenanthrenyl, indanyl, indenyl, and the like. In some embodiments, aryl groups have from 6 to about 20 carbon atoms, from 6 to about 15 carbon atoms, or from 6 to about 10 carbon atoms. In some embodiments, the aryl group is a substituted or unsubstituted phenyl.

As used herein, the term “carbamyl” to a group of formula —C(O)NH₂.

As used herein, the term “carbonyl”, employed alone or in combination with other terms, refers to a —C(═O)— group, which may also be written as C(O).

As used herein, the term “di(C_(n-m-)alkyl)amino” refers to a group of formula —N(alkyl)₂, wherein the two alkyl groups each has, independently, n to m carbon atoms. In some embodiments, each alkyl group independently has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

As used herein, the term “di(C_(n-m-)alkyl)carbamyl” refers to a group of formula —C(O)N(alkyl)₂, wherein the two alkyl groups each has, independently, n to m carbon atoms. In some embodiments, each alkyl group independently has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

As used herein, “halo” refers to F, Cl, Br, or I. In some embodiments, a halo is F, Cl, or Br.

As used herein, the term “C_(n-m) haloalkyl”, employed alone or in combination with other terms, refers to an alkyl group having from one halogen atom to 2s+1 halogen atoms which may be the same or different, where “s” is the number of carbon atoms in the alkyl group, wherein the alkyl group has n to m carbon atoms. In some embodiments, the haloalkyl group is fluorinated only. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

As used herein, “cycloalkyl” refers to non-aromatic cyclic hydrocarbons including cyclized alkyl and/or alkenyl groups. Cycloalkyl groups can include mono- or polycyclic (e.g., having 2, 3 or 4 fused rings) groups and spirocycles. Cycloalkyl groups can have 3, 4, 5, 6, 7, 8, 9, or 10 ring-forming carbons (C₃₋₁₀). Ring-forming carbon atoms of a cycloalkyl group can be optionally substituted by oxo or sulfido (e.g., C(O) or C(S)). Cycloalkyl groups also include cycloalkylidenes. Example cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclopentenyl, cyclohexenyl, cyclohexadienyl, cycloheptatrienyl, norbornyl, norpinyl, norcarnyl, and the like. In some embodiments, cycloalkyl is cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclopentyl, or adamantyl. In some embodiments, the cycloalkyl has 5-10 ring-forming carbon atoms. In some embodiments, cycloalkyl is adamantyl. Also included in the definition of cycloalkyl are moieties that have one or more aromatic rings fused (i.e., having a bond in common with) to the cycloalkyl ring, for example, benzo or thienyl derivatives of cyclopentane, cyclohexane, and the like. A cycloalkyl group containing a fused aromatic ring can be attached through any ring-forming atom including a ring-forming atom of the fused aromatic ring.

As used herein, “heteroaryl” refers to a monocyclic or polycyclic aromatic heterocycle having at least one heteroatom ring member selected from sulfur, oxygen, and nitrogen. In some embodiments, the heteroaryl ring has 1, 2, 3, or 4 heteroatom ring members independently selected from nitrogen, sulfur and oxygen. In some embodiments, any ring-forming N in a heteroaryl moiety can be an N-oxide. In some embodiments, the heteroaryl has 5-13 ring atoms and 1, 2, 3 or 4 heteroatom ring members independently selected from nitrogen, sulfur and oxygen. For example, a five-membered heteroaryl ring is a heteroaryl with a ring having five ring atoms wherein one or more (e.g., 1, 2, or 3) ring atoms are independently selected from N, O, and S. Exemplary five-membered ring heteroaryls are thienyl, furyl, pyrrolyl, imidazolyl, thiazolyl, oxazolyl, pyrazolyl, isothiazolyl, isoxazolyl, 1,2,3-triazolyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-triazolyl, 1,2,4-thiadiazolyl, 1,2,4-oxadiazolyl, 1,3,4-triazolyl, 1,3,4-thiadiazolyl, and 1,3,4-oxadiazolyl. A six-membered heteroaryl ring, for example, is a heteroaryl with a ring having six ring atoms wherein one or more (e.g., 1, 2, or 3) ring atoms are independently selected from N, O, and S. Exemplary six-membered ring heteroaryls are pyridyl, pyrazinyl, pyrimidinyl, triazinyl and pyridazinyl.

As used herein, “heterocycloalkyl” refers to non-aromatic monocyclic or polycyclic heterocycles having one or more ring-forming heteroatoms selected from O, N, or S. Included in heterocycloalkyl are monocyclic 4-10 membered heterocycloalkyl groups. Heterocycloalkyl groups can also include spirocycles. Example heterocycloalkyl groups include pyrrolidin-2-one, 1,3-isoxazolidin-2-one, pyranyl, tetrahydropuran, oxetanyl, azetidinyl, morpholino, thiomorpholino, piperazinyl, tetrahydrofuranyl, tetrahydrothienyl, piperidinyl, pyrrolidinyl, isoxazolidinyl, isothiazolidinyl, pyrazolidinyl, oxazolidinyl, thiazolidinyl, imidazolidinyl, azepanyl, benzazapene, and the like. Ring-forming carbon atoms and heteroatoms of a heterocycloalkyl group can be optionally substituted by oxo or sulfido (e.g., C(O), S(O), C(S), or S(O)₂, etc.). The heterocycloalkyl group can be attached through a ring-forming carbon atom or a ring-forming heteroatom. In some embodiments, the heterocycloalkyl group contains 0 to 3 double bonds. In some embodiments, the heterocycloalkyl group contains 0 to 2 double bonds. Also included in the definition of heterocycloalkyl are moieties that have one or more aromatic rings fused (i.e., having a bond in common with) to the cycloalkyl ring, for example, benzo or thienyl derivatives of piperidine, morpholine, azepine, etc. A heterocycloalkyl group containing a fused aromatic ring can be attached through any ring-forming atom including a ring-forming atom of the fused aromatic ring. In some embodiments, the heterocycloalkyl has 4-10, 4-7 or 4-6 ring atoms with 1, 2, or 3 ring heteroatoms independently selected from nitrogen, oxygen, or sulfur and having one or more oxidized ring members.

At certain places, the definitions or embodiments refer to specific rings (e.g., an azetidine ring, a pyridine ring, etc.). Unless otherwise indicated, these rings can be attached to any ring member provided that the valency of the atom is not exceeded. For example, an azetidine ring may be attached at any position of the ring, whereas a pyridin-3-yl ring is attached at the 3-position.

The term “compound” as used herein is meant to include all stereoisomers, geometric isomers, tautomers, and isotopes of the structures depicted. Compounds herein identified by name or structure as one particular tautomeric form are intended to include other tautomeric forms unless otherwise specified.

Compounds provided herein also include tautomeric forms. Tautomeric forms result from the swapping of a single bond with an adjacent double bond together with the concomitant migration of a proton. Tautomeric forms include prototropic tautomers which are isomeric protonation states having the same empirical formula and total charge. Example prototropic tautomers include ketone—enol pairs, amide—imidic acid pairs, lactam—lactim pairs, enamine—imine pairs, and annular forms where a proton can occupy two or more positions of a heterocyclic system, for example, 1H- and 3H-imidazole, 1H-, 2H- and 4H-1,2,4-triazole, 1H- and 2H-isoindole, and 1H- and 2H-pyrazole. Tautomeric forms can be in equilibrium or sterically locked into one form by appropriate substitution.

All compounds, and pharmaceutically acceptable salts thereof, can be found together with other substances such as water and solvents (e.g. hydrates and solvates) or can be isolated.

In some embodiments, preparation of compounds can involve the addition of acids or bases to affect, for example, catalysis of a desired reaction or formation of salt forms such as acid addition salts.

Example acids can be inorganic or organic acids and include, but are not limited to, strong and weak acids. Some example acids include hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, p-toluenesulfonic acid, 4-nitrobenzoic acid, methanesulfonic acid, benzenesulfonic acid, trifluoroacetic acid, and nitric acid. Some weak acids include, but are not limited to acetic acid, propionic acid, butanoic acid, benzoic acid, tartaric acid, pentanoic acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, and decanoic acid.

Example bases include lithium hydroxide, sodium hydroxide, potassium hydroxide, lithium carbonate, sodium carbonate, potassium carbonate, and sodium bicarbonate. Some example strong bases include, but are not limited to, hydroxide, alkoxides, metal amides, metal hydrides, metal dialkylamides and arylamines, wherein; alkoxides include lithium, sodium and potassium salts of methyl, ethyl and t-butyl oxides; metal amides include sodium amide, potassium amide and lithium amide; metal hydrides include sodium hydride, potassium hydride and lithium hydride; and metal dialkylamides include lithium, sodium, and potassium salts of methyl, ethyl, n-propyl, iso-propyl, n-butyl, tert-butyl, trimethylsilyl and cyclohexyl substituted amides.

In some embodiments, the compounds provided herein, or salts thereof, are substantially isolated. By “substantially isolated” is meant that the compound is at least partially or substantially separated from the environment in which it was formed or detected. Partial separation can include, for example, a composition enriched in the compounds provided herein. Substantial separation can include compositions containing at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% by weight of the compounds provided herein, or salt thereof. Methods for isolating compounds and their salts are routine in the art.

The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The present application also includes pharmaceutically acceptable salts of the compounds described herein. As used herein, “pharmaceutically acceptable salts” refers to derivatives of the disclosed compounds wherein the parent compound is modified by converting an existing acid or base moiety to its salt form. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The pharmaceutically acceptable salts of the present application include the conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of the present application can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, non-aqueous media like ether, ethyl acetate, alcohols (e.g., methanol, ethanol, iso-propanol, or butanol) or acetonitrile (MeCN) are preferred. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418 and Journal of Pharmaceutical Science, 66, 2 (1977). Conventional methods for preparing salt forms are described, for example, in Handbook of Pharmaceutical Salts: Properties, Selection, and Use, Wiley-VCH, 2002.

EXAMPLES

The invention will be described in greater detail by way of specific examples. The following examples are offered for illustrative purposes, and are not intended to limit the invention in any manner. Those of skill in the art will readily recognize a variety of non-critical parameters which can be changed or modified to yield essentially the same results.

Example 1 High Throughput Screening Assay

A large scale in vivo high throughput screen (HTS) was performed to identify small compounds that stimulate lipid production and accumulation using the model organism Chlamydomonas reinhardtii. The HTS employed a 384-well microplate format in which cells were allowed to grow in the presence of the test compounds at 10 μM final concentration for 72 hours. Accumulation of intracellular lipids was assessed using the lipophilic dye Nile Red to select high lipid producers and growth was monitored at OD600 to select compounds that did not eliminate cellular growth.

The screening library included 43,736 compounds (ChemBridge, Corp). A total of 367 active compounds were identified that stimulated lipid accumulation to >2.5-fold and did not affect cell viability. A sub-set of compounds were retested using an 8-point dose response assay (0.25-30 Compounds were further assessed visually using a Nikon Ti-inverted microscope (60×) to reconfirm lipid droplet accumulation was induced. The EC₅₀ (μM) values and microscopy images of compounds 1, 62, and 136 are shown in FIG. 1. Treatment of the cells with compounds 1, 62, and 136 does not disrupt major organelles including nucleus, endoplasmic reticulum, and chloroplast (FIG. 1).

Table 7 shows results of the 8-point dose response assay.

TABLE 7 Dose Response Assay 30 15 10 5 2.5 1.25 0.625 0.25 μM μM μM μM μM μM μM μM 1 21.1 15.48 4.46 9.79 3.08 1.43 1.01 0.84 2 2.11 1.34 1.53 1.1 0.99 0.94 1.01 0.96 3 10.85 3.96 2.39 2.15 1.69 1.19 0.99 0.92 4 4.75 10.37 10.31 10.65 11.42 10.05 1.8 0.94 5 2.81 1.96 2.07 1.28 0.91 0.80 0.85 0.97 6 15.14 17.67 7.75 9.91 3.31 1.99 1.51 0.94 7 7.55 9.14 7.39 10.8 9.26 8.75 8.84 7 8 5.94 13.62 9.43 12.88 3.48 1.57 1.07 1.02 9 9.66 11.32 6.34 9.14 5.29 4.88 7.65 3.47 11 1.78 2.28 2.78 1.46 0.93 0.97 1.12 1.02 12 7.51 5.88 3.39 5.03 1.36 0.85 0.78 1.19 13 6.01 4.05 7.68 7.65 10.91 9.67 2.46 1.17 14 13.04 16.13 8.91 11.61 4.45 11.44 3.73 1.39 15 14.89 7.24 10.5 10.92 3.11 1.39 1.02 0.92 16 2.95 2.7 2.78 11.47 10.51 2.5 1.36 0.99 17 1.67 1.04 0.92 1.07 0.89 0.89 1.03 1.09 18 1.29 1.31 1.19 1.17 1.13 0.89 0.97 1.01 19 7.47 15.55 9.8 10.55 9.13 3.4 2.2 2.39 20 5.51 4.02 2.85 10.58 13.55 11.38 2.97 1.15 21 2.91 5.67 4.65 9.6 7.76 3.64 4.2 1.63 22 4.28 8.64 7.47 12.27 7.88 6.68 2.51 2.53 23 1.15 1.43 1.1 1.04 0.78 0.79 1 1.02 24 1.25 1.19 1.18 0.99 0.97 0.84 0.87 1.13 25 1.34 1.33 1 1 1.02 0.99 1.13 1.15 26 2.94 4.06 6.43 12.5 3.9 1.03 0.97 1.08 27 5.68 3.65 3.57 12.11 14.97 1.86 1.22 1.17 28 13.49 19.07 7.39 11.25 9.91 4.38 5.73 3.06 29 17.22 20.41 7.81 8.14 2.38 2.19 1.43 0.81 30 1.73 1.37 0.82 0.99 0.84 1.16 1.05 1.15 31 17.01 15.24 3.34 4.4 3.61 1.68 0.92 0.86 32 7.09 8.09 3.63 4.84 2.51 0.95 0.78 0.93 33 7.79 10.01 4 1.58 1.18 0.81 0.89 0.82 34 2.35 3.78 3.65 10.44 3.5 1.84 1.01 0.93 35 2.51 2.5 2.65 1.91 1.96 1.23 1.49 1.09 36 5.31 8.49 2.72 3.47 1.89 1.95 0.94 0.93 37 1.15 1.16 1.76 1.93 1.85 1.39 0.97 1.04 38 2 2.61 2.94 2.64 2.23 1.23 1.38 1.06 39 1.38 1.57 0.88 1.18 1.18 0.99 1.13 1.17 40 15.88 17.74 4.07 3.53 3.94 2.25 1.26 1.06 41 7.43 7.85 8.37 15.71 3.33 1 0.99 1.01 42 7.26 6.28 4.24 11.32 6 10.94 10.42 2.01 43 12.59 14.28 5.78 8.39 7.57 9.14 9.13 2.35 44 10.81 13.88 2.68 5.1 1.82 0.95 1.01 1.05 45 1.56 2.06 2.02 3.61 2.3 1.57 0.99 0.93 46 1.78 2.63 2.75 2.31 1.61 0.94 0.96 1.23 47 1.19 1.27 1.37 0.95 1.3 0.91 0.81 0.90 48 6.94 10.55 4.43 8.42 1.69 0.75 0.66 0.80 49 3.07 8.51 8.98 10.46 2.42 1.94 0.93 1.15 50 13.02 8.62 7.3 10.74 3.25 0.90 1.19 1.1 51 11.93 13.34 6.57 9.13 3.34 3.47 3.9 1.09 52 11.4 10.15 9.09 7.75 6.48 0.91 0.92 1.12 53 2.49 1.61 1.57 1.42 1.1 1.13 1.38 1.06 54 1.58 1.78 1.73 2.79 2.32 2.13 1.53 1.05 55 2.77 2.26 2.04 2.23 2.27 2.01 1.77 1.3 56 1.92 1.74 2.71 2.58 1.8 1.34 1.2 1.2 57 1.87 1.15 1.11 1.28 0.90 1.01 1.06 0.99 58 1.56 1.42 1.15 1.22 0.90 1.14 0.94 1.06 59 3.84 3.04 2.52 2.54 2.18 2.02 1.91 1.28 60 1.65 1.35 1.4 1.22 1.1 1 1.07 1.07 61 1.14 1.24 0.91 1.04 0.95 0.92 0.96 0.90 62 21.07 19.19 8.95 10.44 8.17 3.11 3.12 1.05 63 21.78 20.48 10.18 9.63 3.92 2.53 1.61 0.98 64 7.98 3.48 7.63 10.61 10.14 7.72 11.51 5.81 65 8.07 11.17 8.09 9.72 5.42 3.84 2.04 1.03 66 5.72 4.89 5.36 9.73 12.08 9.49 4.1 1.31 67 3.6 6.33 7.75 10.08 13.05 3.09 2.29 1 68 2.03 2.74 1.95 2.1 2.15 1.28 1.05 1.07 69 2.54 3.49 3.49 2.44 3.01 1.29 0.96 1.05 70 11.07 11.05 7.57 8.64 4.4 2.19 1.55 0.87 71 11.83 12.08 8.37 7.95 2.29 1.22 1.15 0.89 72 9.85 10.08 9.28 2.81 2.21 0.99 1.03 1.22 74 2.81 1.96 2.07 1.28 0.91 0.80 0.85 0.97 75 6.95 2.89 2.35 2.25 2.08 1.22 1.02 0.90 76 10.01 11.14 8.88 5.17 10.31 8.1 8.99 2.09 77 4.05 10.93 2.78 2.28 10.74 2.6 1.41 1.06 78 7.85 10.45 9.94 8.34 10.24 1.64 1.41 0.83 79 16.86 15.62 8.1 9.69 6.13 1.83 1.08 0.87 81 18.89 18.42 3.75 3.32 1.97 0.88 0.85 0.91 82 13.39 17.76 8.09 10.46 7.09 8.56 8.43 4.74 83 8.83 15.98 6.89 1.14 1.01 0.76 0.79 0.98 84 1.34 2.19 1.64 2.05 2.5 1 0.89 0.92 86 3.76 3.38 2.71 2.29 2.39 1.69 1.01 0.98 88 4.8 15.2 8.16 10.2 8.96 4.98 2.14 1.04 90 8.7 13.43 6.74 8.56 6.5 2.86 8.53 2.39 91 8.29 16.37 6.76 10.52 11.82 2.82 1.37 0.95 93 8.86 12.09 8.59 7.33 3.6 1.49 0.96 0.97 94 6.35 11.58 9.97 16.62 11.42 10.23 10.57 5.93 95 1.81 1.74 4.03 14.02 10.7 1.78 1.5 1.03 96 11.77 17.34 10.03 10.24 6.91 3.17 1.51 0.87 97 8.76 7.56 3.46 9.14 3.15 3.67 3.14 1.1 98 11.04 10.69 8.92 3.28 3.5 2.25 1.57 1.39 99 4.22 3.37 2.96 1.96 1.4 0.97 0.84 0.98 100 18.12 9.57 3.77 3.04 2.26 1.12 0.95 1 101 1.75 1.43 1.04 1.58 0.88 1.14 1.08 1.03 103 9.86 10.84 5.85 1.17 1.1 0.94 0.85 0.99 104 3.69 3.28 2.81 4.04 5.27 3.96 3.68 1.23 106 9.7 12.36 5.08 6.74 3.68 2.46 2.12 1.02 107 3.06 3.07 2.48 2.31 2.05 1.23 1.01 0.98 109 9.73 12.09 5.27 5.91 6.87 2.89 1.93 1.42 110 12.43 14.61 7.3 3.69 3.61 3.52 1.76 0.89 111 9.13 12.3 6.41 8.78 7.07 8.14 2.59 1.04 112 10.17 7.57 12.3 12.95 3.53 1.3 0.86 0.86 113 3.03 4.24 5.96 11.79 11.03 10.63 2.27 1.15 120 1.02 3.19 2.15 1.07 1.12 0.89 0.64 0.91 121 12.49 11.68 5.87 9.06 1.93 3.47 1.31 0.92 123 1.83 3.09 3.7 4.8 10.38 7.48 4.34 1.86 124 1.38 1.57 0.88 1.18 1.18 0.99 1.13 1.17 129 14.66 14.1 3.85 15.81 12.76 2.18 1.34 0.95 130 7.38 15.13 6.34 4.88 1.03 0.94 0.96 1.13 131 1.87 4.36 9.13 3.8 1.66 1.08 1.02 0.99 132 4.57 5.24 2.62 2.87 1.91 1.16 1.19 1.02 133 3.44 14.41 9.72 12.58 7.4 2.81 2.02 1 134 2.14 1.97 1.31 1.77 1.32 0.93 1.06 0.97 135 11.19 16.64 10.02 9.84 2.47 1.09 0.88 0.86 136 19.83 18.82 8.7 12.81 3.51 1.82 1.32 0.88 137 4.85 12.33 7.15 10.2 2.77 3.13 1.91 1.09 138 12.1 16.27 9.72 9.85 2.75 3.51 2.28 1.09 139 2.8 6.14 3.04 2.27 2.68 0.86 1.09 1.18 140 7.63 5.97 4.8 3.05 1.26 1.1 1.33 0.98 141 4.1 3.53 4.5 13.19 11.63 9.16 3.57 1.39 142 7.36 4 6.3 6.53 10.4 10.17 13.32 9.79 143 14.27 17.97 9.99 9.45 9.14 2.68 1.39 0.85 144 19.74 22.79 11.58 12.44 10.13 4.03 1.64 0.86 145 11.88 13.65 7.27 8.15 7.77 9.73 2.56 0.81 146 6.76 5.68 6.61 4.14 4.38 1.93 1.3 1.01 147 2.84 3.72 5.17 9.42 11.21 9.71 11.86 2.58 148 1.45 1.65 1.39 1.63 1.32 0.88 1.04 0.94 149 8.77 16.41 3.82 5.3 3.45 1.18 0.84 0.84

Example 2 Cellular Growth

Chlamydomonas reinhardtii cells were grown in the presence of DMSO (control) or 30 82 M of test compound for 72 hours. At the indicated times 200 μL cells were removed from each well and optical density and nile red fluorescence were measured. Results of the algal cell growth study are shown in FIG. 2A-2C.

Example 3 Lipid Accumulation

Representative compounds provided herein were shown to increase lipid accumulation using lipophilic dye Nile Red at concentrations ranging from 0.5 μM to 15.0 μM in Chlamydomonas reinhardtii cells, as shown in FIG. 3. Lipid accumulation over a period of 72 hours was also measured (30 μM test compound) using lipophilic dye Nile Red, as shown in FIG. 4. The increase of specific fatty acids and triglyercide accumulation was also demonstrated using compounds 1, 62, and 136 as shown in FIGS. 5-6 (compounds tested 30 μM for fatty acid assay and 5 μM for triglyceride assay). FIGS. 12A-12C show representative images of C. reinhardtii cultures that were treated with 10 μM of representative test compounds of Formula Ia, Formula IIa, and Formula IIIa. The corresponding lipid accumulation was visualized using confocal microscopy after 72 hours in culture.

Example 4 Cellular Chlorophyll, Protein, and Carbohydrate Levels

Compounds 1, 62, and 136 (30 μM) did not significantly alter levels of chlorophyll A (Chl A), chlorophyll B (Chl B), or carotenoid levels in Chlamydomonas reinhardtii cells, as shown in FIG. 7. Further, compounds 1 and 136 did not significantly affect the levels of proteins required for photosynthesis (e.g., Rubisco large sub unit, Rubisco small sub unit, PSa D Photosystem I, PSb A Photosystem II, phosphoribulokinase, and histone, and compound 62 exhibited minor effects on the same proteins, as shown in FIG. 8. All three test compounds (30 μM) increased carbohydrate levels in Chlamydomonas reinhardtii cells, indicating stimulation of starch storage in addition to lipid storage, as shown in FIG. 9. None of the test compounds significantly affected total protein levels, as shown in FIG. 10.

Example 5 Lipid Accumulation in Algal Cells

Compounds 1, 62, and 136 were assayed in algal species Chlamydomonas reinhardtii CC125, Chlorella sorokiniana UTEX 1230, Chlorella vulgaris UTEX 395, and Tetrachlorella alterns UTEX 2453 at concentrations ranging from 0.63 μM to 50 μM. All three test compounds stimulated lipid accumulation in each of the algal species, as shown in FIG. 11A-11C.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. 

1. A method of increasing lipid accumulation in a freshwater algal cell, comprising contacting the cell with an effective amount of a compound of Formula Ia:

or a salt thereof, wherein: L is selected from the group consisting of, C₁₋₄ alkylene, and —(C₁₋₄ alkylene)-NR^(L)-(C₁₋₄ alkylene); R^(L) is selected from the group consisting of H and methyl; R³ is H; R⁴ is; Cy¹ is selected from the group consisting of phenyl; R^(Cy) is selected from the group consisting of halo, C₁₋₄ alkyl, C₁₋₄ alkoxy, phenyl, —(C₁₋₄ alkylene)-(phenyl), C₃₋₁₀ cycloalkyl, and —(C₁₋₄ alkylene)-(C₃₋₁₀ cyloalkyl), wherein each phenyl and the phenyl group of —(C₁₋₄ alkylene)-(phenyl) is optionally substituted by 1 or 2 substituents independently selected from the group consisting of OH, halo, C₁₋₄ alkyl, and C₁₋₄ alkoxy; n is 0 or 1; and p is 0, 1, 2, or 3; or a compound of Formula Ila:

or a salt thereof, wherein: L¹ is selected from the group consisting of a bond and C₁₋₄ alkylene: R^(1a) is selected from the group consisting of, C₅₋₁₀ cycloalkyl, 8-10 membered heteroaryl, phenyl, and 10-13 membered heteroaryl, wherein the C₅₋₁₀ cycloalkyl and phenyl are each optionally substituted by 1, 2, or 3 substituents independently selected from the group consisting of halo, OH, NO₂, C₁₋₄ alkyl, and C₁₋₄ alkoxy, and wherein the 10-13 membered heteroaryl is optionally substituted by one halo group; R² is selected from the group consisting of H, halo, OH, NO₂, C₁₋₄ alkyl, and C₁₋₄ alkoxy; R³ is selected from the group consisting of H, halo, and C₁₋₄ alkoxy; R⁴ is selected from the group consisting of H, OH, and C₁₋₄ alkoxy; R⁵ is selected from the group consisting of H, halo, and C₁₋₄ alkoxy; and R⁶ is; or a compound of Formula IIIa:

or a salt thereof, wherein: L¹ is selected from the group consisting of, NH and N(C₁₋₄ alkyl); R^(1a) is selected from the group consisting of H, C₁₋₄ alkyl, C₂₋₄ alkenyl, C₁₋₄ cyanoalkylene, NH(C₁₋₄ alkyl), and NH(C₂₋₄ alkenyl); L² is; and R^(2a) is selected from the group consisting of H, C₁₋₄ hydroxyalkyl, —SO₂(C₁₋₄ alkyl).
 2. The method of claim 1, wherein the compound is a compound of Formula Ia.
 3. The method of claim 1, wherein the compound is a compound of Formula IIa.
 4. The method of claim 1, wherein the compound is a compound of Formula IIIa. 5.-6. (canceled)
 7. The method of claim 1, wherein the freshwater algal cell is a Chlorophyceae cell.
 8. The method of claim 1, wherein the freshwater algal cell is selected from the group consisting of a Chlamydomonas cell, a Chlorella cell, a Tetrachlorella cell, and a Nannochloropsis cell. 9.-18. (canceled)
 19. The method of claim 1, wherein the contacting the cell with the compound does not reduce the rate of cellular growth compared to a cell that has not been contacted by the compound. 20.-21. (canceled)
 22. The method of claim 1, wherein the lipid accumulation is increased without exposing the cell to environmental stress.
 23. The method of claim 22, wherein the environmental stress comprises nutrient deprivation. 24.-49. (canceled)
 50. The method of claim 2, wherein L is C₁₋₄ alkylene.
 51. The method of claim 2, wherein each R^(Cy) is an independently selected C₁₋₄ alkoxy.
 52. The method of claim 2, wherein p is 1 or
 2. 53. The method of claim 3, wherein L¹ is a bond.
 54. The method of claim 3, wherein R^(1a) is phenyl substituted by C₁₋₄ alkoxy.
 55. The method of claim 3, wherein R² is OH.
 56. The method of claim 3, wherein R³, R⁴, and R⁵ are each H.
 57. The method of claim 4, wherein L¹ is NH.
 58. The method of claim 4, wherein R^(1a) is C₁₋₄ cyanoalkylene.
 59. The method of claim 4, wherein R^(2a) is H.
 60. The method of claim 1, wherein the compound is selected from the group consisting of:

or a salt thereof. 